Pretargeting protocols for the enhanced localization of cytotoxins to target sites and cytotoxic combinations useful therefore

ABSTRACT

Methods for targeting cytotoxins to target sites by administration of a combination of conjugates are provided. Novel cytotoxic combinations for use in such methods are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation, of application Ser. No. 08/163,188,filed Dec. 7, 1993, now abandoned, in turm a CIP of PCT/US93/05406,filed Jun. 7, 1993, in turn a CIP of application Ser. No. 07/995,381,filed Dec. 23, 1992 now abandoned, in turn a CIP of application Ser. No.07/895,588, filed Jun. 9, 1992 now U.S. Pat. No. 5,288,342.

TECHNICAL FIELD

The present invention relates to methods, compounds, compositions andkits useful for delivering to a target site a targeting moiety that isconjugated to one member of a ligand/anti-ligand pair. Afterlocalization and clearance of the targeting moiety conjugate, direct orindirect binding of a diagnostic or therapeutic agent conjugate at thetarget site occurs.

BACKGROUND OF THE INVENTION

Conventional cancer therapy is plagued by two problems. The generallyattainable targeting ratio (ratio of administered dose localizing totumor versus administered dose circulating in blood or ratio ofadministered dose localizing to tumor versus administered dose migratingto bone marrow) is low. Also, the absolute dose of radiation ortherapeutic agent delivered to the tumor is insufficient in many casesto elicit a significant tumor response. Improvement in targeting ratioor absolute dose to tumor is sought.

SUMMARY OF THE INVENTION

The present invention is directed to diagnostic and therapeuticpretargeting methods, moieties useful therein and methods of makingthose moieties. Such pretargeting methods are characterized by animproved targeting ratio or increased absolute dose to the target cellsites in comparison to conventional cancer therapy.

The present invention provides for effective delivery of cytotoxicactive agents such as toxins, bacterial toxins and fungal metabolites,including highly toxic moieties such as palytoxins. The decoupling ofthe pharmacokinetics of the targeting moiety (generally slow) and theactive agent (generally rapid for low molecular weight active agents andgenerally somewhat slower for higher molecular weight active agents whenadministered alone) and the high affinity binding of ligand-anti-ligandpairs provide for this improvement. When the active agents or activeagent-ligand or active agent-anti-ligand conjugates to be administeredare not themselves generally rapidly cleared (preferably via the renalpathway), conjugates containing such active agents are constructed toimpart relatively rapid, and preferably renal, clearance thereto orlower, therapeutically effective, doses of active agent areadministered. Thus, the protocol recipient's non-target tissue does notsuffer prolonged exposure to the active agent.

The present invention provides two-step and three-step pretargetingmethods, employing the steps set forth below:

administering to the recipient a first conjugate including an antibodytargeting moiety of a first antibody species having a first pattern ofcross-reactivity and a member of a ligand-anti-ligand binding pair; and

administering to the recipient one or more additional targetingconjugates, each such conjugate including an antibody targeting moietyof a different species from the species of the first conjugate and eachother having a substantially non-overlapping pattern of cross reactivityfrom that of other additional targeting conjugates and from each otherand from the first pattern of cross-reactivity and the member of theligand-anti-ligand pair bound to the first conjugate.

In the practice of these aspects of the present invention, target siteaccretion of active agent conjugate receptor (i.e., the ligand oranti-ligand conjugated to the first antibody species and additionaltargeting antibody species) is improved, because each antibody speciesrecognizes a different epitope associated with the target site. Thisalternative epitope approach provides a target site that is more denselypopulated with the anti-ligand or ligand antigen to which thesubsequently administered active agent-containing conjugate may bind viahigh affinity ligand-anti-ligand interactions. This increased targetsite antigen density facilitates increased active agent accretionthereto.

The present invention also provides pretargeting photodynamic therapyprotocols as set forth below.

The two-step approach involves:

administering to the recipient a first conjugate comprising a targetingmoiety and a member of a ligand-anti-ligand binding pair, wherein thefirst conjugate localizes to a target site;

optionally administering to the recipient a clearing agent capable ofdirecting the clearance of circulating conjugate from the recipient oroptionally treating the recipient with a clearing device or analternative clearing procedure to substantially remove circulatingconjugate from the recipient; and

administering to the recipient a second conjugate comprising aphotosensitizing agent and a ligand/anti-ligand binding pair member,wherein the second conjugate binding pair member is complementary tothat of the first conjugate and wherein the photosensitizing agent orthe second conjugate is chemically modified to induce rapid and,preferably, renal clearance thereof from the recipient.

One alternative to the optional clearance step set forth above is simplyto allow an amount of time to pass that is sufficient to permit therecipient's native clearance mechanisms to substantially removecirculating first conjugate.

The three-step approach involves:

administering to the recipient a first conjugate comprising a targetingmoiety and a ligand, wherein the targeting moiety-ligand conjugatelocalizes to a target site;

administering to the recipient an anti-ligand; and

administering to the recipient a second conjugate comprising the ligandand a photosensitive agent, wherein the photosensitizing agent or thesecond conjugate is chemically modified to induce rapid and, preferably,renal clearance thereof from the recipient and wherein second conjugatelocalization at the target site is enhanced as a result of priorlocalization of the first conjugate.

While the two-step and three-step pretargeting methods of the presentinvention may be conducted despite the presence of recipient endogenousbiotin, the present invention also provides methods of decreasing theendogenous biotin level or the impact thereof. One method is tooverwhelm the endogenous biotin with a high dose of targetingmoiety-streptavidin or -avidin conjugate. Another method is apretreatment with an amount of avidin sufficient to bind substantiallyall of a recipient's endogenous biotin. In conducting this method,avidin may be administered intravenously, orally or by enema.Alternatively, the recipient may be placed on a biotin-free diet priorto conducting a two-step or three-step pretargeting protocol. Anothermethod to address endogenous biotin employs oral, non-absorbableantibiotics.

Cytokines, such as interleukins (e.g., IL-2 and IL-4), colonystimulating factors (e.g., GM-CSF), interferons, (e.g.,interferon-gamma), and tumor necrosis factor (TNF), may be employed asanti-tumor active agents in the practice of two-step or three-steppretargeting protocols of the present invention. In addition, thepretargeting protocols of the present invention have applications withrespect to additional conditions. Immunosuppressive cytokines, such asTGF-beta, may be employed, for example, in the treatment of autoimmunediseases; such as rheumatoid arthritis, systemic lupus erythematosus,insulin-dependent diabetes mellitus, multiple sclerosis, pulmonaryfibrosis and the like; tissue transplantation facilitation in liver andkidney tissues, for example; obviation or prevention ofgraft-versus-host reaction; and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates blood clearance of biotinylated antibody followingintravenous administration of avidin.

FIG. 2 depicts radiorhenium tumor uptake in a three-step pretargetingprotocol, as compared to administration of radiolabeled antibody(conventional means involving antibody that is covalently linked tochelated radiorhenium).

FIG. 3 depicts the tumor uptake profile of NR-LU-10-streptavidinconjugate (LU-10-StrAv) in comparison to a control profile of nativeNR-LU-10 whole antibody,

FIG. 4 depicts the tumor uptake and blood clearance profiles ofNR-LU-10-streptavidin conjugate.

FIG. 5 depicts the rapid clearance from the blood of asialoorosomucoidin comparison with orosomucoid in terms of percent injected dose ofI-125-labeled protein.

FIG. 6 depicts the 5 minute limited biodistribution of asialoorosomucoidin comparison with orosomucoid in terms of percent injected dose ofI-125-labeled protein.

FIG. 7 depicts NR-LU-10-streptavidin conjugate blood clearance uponadministration of three controls (∘,,▪) and two doses of a clearingagent (¤,□) at 25 hours post-conjugate administration.

FIG. 8 shows limited biodistribution data for LU-10-StrAv conjugate uponadministration of three controls (Groups 1, 2 and 5) and two doses ofclearing agent (Groups 3 and 4) at two hours post-clearing agentadministration.

FIG. 9 depicts NR-LU-10-streptavidin conjugate serum biotin bindingcapability at 2 hours post-clearing agent administration.

FIG. 10 depicts NR-LU-10-streptavidin conjugate blood clearance overtime upon administration of a control (◯) and three doses of a clearingagent (∇, Δ,□) at 24 hours post-conjugate administration.

FIG. 11A depicts the blood clearance of LU-10-StrAv conjugate uponadministration of a control (PBS) and three doses (50, 20 and 10 μg) ofclearing agent at two hours post-clearing agent administration.

FIG. 11B depicts LU-10-StrAv conjugate serum biotin binding capabilityupon administration of a control (PBS) and three doses (50, 20 and 10μg) of clearing agent at two hours post-clearing agent administration.

FIG. 12 depicts a three-step pretargeting approach.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention, it may be helpful to set forthdefinitions of certain terms to be used within the disclosure.

Targeting moiety: A molecule that binds to a defined population ofcells. The targeting moiety may bind a receptor, an oligonucleotide, anenzymatic substrate, an antigenic determinant, or other binding sitepresent on or in the target cell population. Antibody is used throughoutthe specification as a prototypical example of a targeting moiety.Antibody fragments and small peptide sequences capable of recognizingexpressed antigen are also contemplated targeting moieties within thepresent invention. Tumor is used as a prototypical example of a targetin describing the present invention.

Ligand/anti-ligand pair: A complementary/anti-complementary set ofmolecules that demonstrate specific binding, generally of relativelyhigh affinity. Exemplary ligand/anti-ligand pairs include zinc fingerprotein/dsDNA fragment, enzyme/inhibitor, hapten/antibody,lectin/carbohydrate, ligand/receptor, and biotin/avidin. Lower molecularweight forms of the ligand or anti-ligand molecules that bind withcomplementary anti-ligands or ligands are also contemplated by thepresent invention. Biotin/avidin is used throughout the specification asa prototypical example of a ligand/anti-ligand pair.

Anti-ligand: As defined herein, an "anti-ligand" demonstrates highaffinity, and preferably, multivalent binding of the complementaryligand. Preferably, the anti-ligand is large enough to avoid rapid renalclearance, and contains sufficient multivalency to accomplishcrosslinking and aggregation of targeting moiety-ligand conjugates.Univalent anti-ligands are also contemplated by the present invention.Anti-ligands of the present invention may exhibit or be derivatized toexhibit structural features that direct the uptake thereof, e.g.,galactose residues that direct liver uptake. Avidin and streptavidin areused herein as prototypical anti-ligands.

Avidin: As defined herein, "avidin" includes avidin, streptavidin andderivatives and analogs thereof that are capable of high affinity,multivalent or univalent binding of biotin.

Ligand: As defined herein, a "ligand" is a relatively small, solublemolecule that exhibits rapid serum, blood and/or whole body clearancewhen administered intravenously in an animal or human. Biotin is used asthe prototypical ligand.

Active Agent: A diagnostic or therapeutic agent ("the payload"),including radionuclides, drugs, anti-tumor agents, toxins and the like.Radionuclide therapeutic agents are used as prototypical active agents.

N_(x) S_(y) Chelates: As defined herein, the term "N_(x) S_(y) chelates"includes buoy chelators that are capable of (i) coordinately binding ametal or radiometal and (ii) covalently attaching to a targeting moiety,ligand or anti-ligand. Particularly preferred N_(x) S_(y) chelates haveN₂ S₂ and N₃ S cores. Exemplary N_(x) S_(y) chelates are described inFritzberg et al., Proc. Natl. Acad. Sci. USA 85:4024-29, 1988; in Weberet al., Bioconj. Chem. 1:431-37, 1990; and in the references citedtherein, for instance.

Pretargeting: As defined herein, pretargeting involves target sitelocalization of a targeting moiety that is conjugated with one member ofa ligand/anti-ligand pair; after a time period sufficient for optimaltarget-to-non-target accumulation of this targeting moiety conjugate,active agent conjugated to the opposite member of the ligand/anti-ligandpair is administered and is bound (directly or indirectly) to thetargeting moiety conjugate at the target site (two-step pretargeting).Three-step and other related methods described herein are alsoencompassed.

Clearing Agent: An agent capable of binding, complexing or otherwiseassociating with an administered moiety (e.g., targeting moiety-ligand,targeting moiety-anti-ligand or anti-ligand alone) present in therecipient's circulation, thereby facilitating circulating moietyclearance from the recipient's body, removal from blood circulation, orinactivation thereof in circulation. The clearing agent is preferablycharacterized by physical properties, such as size, charge,configuration or a combination thereof, that limit clearing agent accessto the population of target cells recognized by a targeting moiety usedin the same treatment protocol as the clearing agent.

Target Cell Retention: The amount of time that a radionuclide or othertherapeutic agent remains at the target cell surface or within thetarget cell. Catabolism of conjugates or molecules containing suchtherapeutic agents appears to be primarily responsible for the loss oftarget cell retention.

Conjugate: A conjugate encompasses chemical conjugates (covalently ornon-covalently bound), fusion proteins and the like.

A recognized disadvantage associated with in vivo administration oftargeting moiety-radioisotopic conjugates for imaging or therapy islocalization of the attached radioactive agent at both non-target andtarget sites. Until the administered radiolabeled conjugate clears fromthe circulation, normal organs and tissues are transitorily exposed tothe attached radioactive agent. For instance, radiolabeled wholeantibodies that are administered in vivo exhibit relatively slow bloodclearance; maximum target site localization generally occurs 1-3 dayspost-administration. Generally, the longer the clearance time of theconjugate from the circulation, the greater the radioexposure ofnon-target organs.

These characteristics are particularly problematic with humanradioimmunotherapy. In human clinical trials, the long circulatinghalf-life of radioisotope bound to whole antibody causes relativelylarge doses of radiation to be delivered to the whole body. Inparticular, the bone marrow, which is very radiosensitive, is thedose-limiting organ of non-specific toxicity.

In order to decrease radioisotope exposure of non-target tissue,potential targeting moieties generally have been screened to identifythose that display minimal non-target reactivity, while retaining targetspecificity and reactivity. By reducing non-target exposure (and adversenon-target localization and/or toxicity), increased doses of aradiotherapeutic conjugate may be administered; moreover, decreasednon-target accumulation of a radiodiagnostic conjugate leads to improvedcontrast between background and target.

Therapeutic drugs, administered alone or as targeted conjugates, areaccompanied by similar disadvantages. Again, the goal is administrationof the highest possible concentration of drug (to maximize exposure oftarget tissue), while remaining below the threshold of unacceptablenormal organ toxicity (due to non-target tissue exposure). Unlikeradioisotopes, however, therapeutic drugs need to be taken into a targetcell to exert a cytotoxic effect. In the case of targetingmoiety-therapeutic drug conjugates, it would be advantageous to combinethe relative target specificity of a targeting moiety with a means forenhanced target cell internalization of the targeting moiety-drugconjugate,

In contrast, enhanced target cell internalization is disadvantageous ifone administers diagnostic agent-targeting moiety conjugates.Internalization of diagnostic conjugates results in cellular catabolismand degradation of the conjugate, Upon degradation, small adducts of thediagnostic agent or the diagnostic agent per se may be released from thecell, thus eliminating the ability to detect the conjugate in atarget-specific manner.

One method for reducing non-target tissue exposure to a diagnostic ortherapeutic agent involves "pretargeting" the targeting moiety at atarget site, and then subsequently administering a rapidly clearingdiagnostic or therapeutic agent conjugate that is capable of binding tothe "pretargeted" targeting moiety at the target site. A description ofsome embodiments of the protargeting technique may be found in U.S. Pat.No. 4,863,713 (Goodwin et al.).

A typical pretargeting approach ("three-step") is schematically depictedin FIG. 12. Briefly, this three-step pretargeting protocol featuresadministration of an antibody-ligand conjugate, which is allowed tolocalize at a target site and to dilute in the circulation. Subsequentlyadministered anti-ligand binds to the antibody-ligand conjugate andclears unbound antibody-ligand conjugate from the blood. Preferredanti-ligands are large and contain sufficient multivalency to accomplishcrosslinking and aggregation of circulating antibody-ligand conjugates.The clearing by anti-ligand is probably attributable to anti-ligandcrosslinking and/or aggregation of antibody-ligand conjugates that arecirculating in the blood, which leads to complex/aggregate clearance bythe recipient's RES (reticuloendothelial system). Anti-ligand clearanceof this type is preferably accomplished with a multivalent molecule;however, a univalent molecule of sufficient size to be cleared by theRES on its own could also be employed. Alternatively, receptor-basedclearance mechanisms, e.g., Ashwell receptor or hexose residue, such asgalactose or mannose residue, recognition mechanisms, may be responsiblefor anti-ligand clearance. Such clearance mechanisms are less dependentupon the valency of the anti-ligand with respect to the ligand than theRES complex/aggregate clearance mechanisms. It is preferred that theligand-anti-ligand pair displays relatively high affinity binding.

A diagnostic or therapeutic agent-ligand conjugate that exhibits rapidwhole body clearance is then administered. When the circulation bringsthe active agent-ligand conjugate in proximity to the target cell-boundantibody-ligand-anti-ligand complex, anti-ligand binds the circulatingactive agent-ligand conjugate and produces anantibody-ligand:anti-ligand:ligand-active agent "sandwich" at the targetsite. Because the diagnostic or therapeutic agent is attached to arapidly clearing ligand (rather than antibody, antibody fragment orother slowly clearing targeting moiety), this technique promisesdecreased non-target exposure to the active agent.

Alternate pretargeting methods eliminate the step of parenterallyadministering an anti-ligand clearing agent. These "two-step" proceduresfeature targeting moiety-ligand or targeting moiety-anti-ligandadministration, followed by administration of active agent conjugated tothe opposite member of the ligand-anti-ligand pair. As an optional step"1.5" in the two-step pretargeting methods of the present invention, aclearing agent (preferably other than ligand or anti-ligand alone) isadministered to facilitate the clearance of circulating targetingmoiety-containing conjugate.

In the two-step pretargeting approach, the clearing agent preferablydoes not become bound to the target cell population, either directly orthrough the previously administered and target cell bound targetingmoiety-anti-ligand or targeting moiety-ligand conjugate. An example oftwo-step pretargeting involves the use of biotinylated human transferrinas a clearing agent for avidin-targeting moiety conjugate, wherein thesize of the clearing agent results in liver clearance oftransferrin-biotin-circulating avidin-targeting moiety complexes andsubstantially precludes association with the avidin-targeting moietyconjugates bound at target cell sites. (See, Goodwin, D. A., Antibod.Immunoconi. Radiopharm., 4: 427-34, 1991).

The two-step pretargeting approach overcomes certain disadvantagesassociated with the use of a clearing agent in a three-step pretargetedprotocol. More specifically, data obtained in animal models demonstratethat in vivo anti-ligand binding to a pretargeted targetingmoiety-ligand conjugate (i.e., the cell-bound conjugate) removes thetargeting moiety-ligand conjugate from the target cell. One explanationfor the observed phenomenon is that the multivalent anti-ligandcrosslinks targeting moiety-ligand conjugates on the cell surface,thereby initiating or facilitating internalization of the resultantcomplex. The apparent loss of targeting moiety-ligand from the cellmight result from internal degradation of the conjugate and/or releaseof active agent from the conjugate (either at the cell surface orintracellularly). An alternative explanation for the observed phenomenonis that permeability changes in the target cell's membrane allowincreased passive diffusion of any molecule into the target cell. Also,some loss of targeting moiety-ligand may result from alteration in theaffinity by subsequent binding of another moiety to the targetingmoiety-ligand, e.g., anti-idiotype monoclonal antibody binding causesremoval of tumor bound monoclonal antibody.

The present invention recognizes that this phenomenon (apparent loss ofthe targeting moiety-ligand from the target cell) may be used toadvantage with regard to in vivo delivery of therapeutic agentsgenerally, or to drug delivery in particular. For instance, a targetingmoiety may be covalently linked to both ligand and therapeutic agent andadministered to a recipient. Subsequent administration of anti-ligandcrosslinks targeting moiety-ligand-therapeutic agent tripartiteconjugates bound at the surface, inducing internalization of thetripartite conjugate (and thus the active agent). Alternatively,targeting moiety-ligand may be delivered to the target cell surface,followed by administration of anti-ligand-therapeutic agent.

In one aspect of the present invention, a targeting moiety-anti-ligandconjugate is administered in vivo; upon target localization of thetargeting moiety-anti-ligand conjugate (i.e., and clearance of thisconjugate from the circulation), an active agent-ligand conjugate isparenterally administered. This method enhances retention of thetargeting moiety-anti-ligand:ligand-active agent complex at the targetcell (as compared with targeting moiety-ligand:anti-ligand:ligand-activeagent complexes and targeting moiety-ligand:anti-ligand-active agentcomplexes). Although a variety of ligand/anti-ligand pairs may besuitable for use within the claimed invention, a preferredligand/anti-ligand pair is biotin/avidin.

In a second aspect of the invention, radioiodinated biotin and relatedmethods are disclosed. Previously, radioiodinated biotin derivativeswere of high molecular weight and were difficult to characterize. Theradioiodinated biotin described herein is a low molecular weightcompound that has been easily and well characterized.

In a third aspect of the invention, a targeting moiety-ligand conjugateis administered in vivo; upon target localization of the targetingmoiety-ligand conjugate (i.e., and clearance of this conjugate from thecirculation), a drug-anti-ligand conjugate is parenterally administered.This two-step method not only provides pretargeting of the targetingmoiety conjugate, but also induces internalization of the subsequenttargeting moiety-ligand-anti-ligand-drug complex within the target cell.Alternatively, another embodiment provides a three-step protocol thatproduces a targeting moiety-ligand:anti-ligand:ligand-drug complex atthe surface, wherein the ligand-drug conjugate is administeredsimultaneously or within a short period of time after administration ofanti-ligand (i.e., before the targeting moiety-ligand-anti-ligandcomplex has been removed from the target cell surface).

In a fourth aspect of the invention, methods for radiolabeling biotinwith technetium-99m, rhenium-186 and rhenium-188 are disclosed.Previously, biotin derivatives were radiolabeled with indium-111 for usein pretargeted immunoscintigraphy (for instance, Virzi et al., Nucl.Med. Biol. 18:719-26, 1991; Kalofonos et al., J. Nucl. Med. 31: 1791≧96,1990; Paganelli et al., Canc. Res. 51:5960-66, 1991). However, ^(99m) Tcis a particularly preferred radionuclide for immunoscintigraphy due to(i) low cost, (ii) convenient supply and (iii) favorable nuclearproperties. Rhenium-186 displays chelating chemistry very similar to^(99m) Tc, and is considered to be an excellent therapeutic radionuclide(i.e., a 3.7 day half-life and 1.07 MeV maximum particle that is similarto ¹³¹ I). Therefore, the claimed methods for technetium and rheniumradiolabeling of biotin provide numerous advantages.

The "targeting moiety" of the present invention binds to a definedtarget cell population, such as tumor cells. Preferred targetingmoieties useful in this regard include antibody and antibody fragments,peptides, and hormones. Proteins corresponding to known cell surfacereceptors (including low density lipoproteins, transferrin and insulin),fibrinolytic enzymes, anti-HER2, platelet binding proteins such asannexins, and biological response modifiers (including interleukin,interferon, erythropoietin and colony-stimulating factor) are alsopreferred targeting moieties. Also, anti-EGF receptor antibodies, whichinternalize following binding to the receptor and traffic to the nucleusto an extent, are preferred targeting moieties for use in the presentinvention to facilitate delivery of Auger emitters and nucleus bindingdrugs to target cell nuclei. Oligonucleotides, e.g., antisenseoligonucleotides that are complementary to portions of target cellnucleic acids (DNA or RNA), are also useful as targeting moieties in thepractice of the present invention. Oligonucleotides binding to cellsurfaces are also useful. Analogs of the above-listed targeting moietiesthat retain the capacity to bind to a defined target cell population mayalso be used within the claimed invention. In addition, synthetictargeting moieties may be designed.

Functional equivalents of the aforementioned molecules are also usefulas targeting moieties of the present invention. One targeting moietyfunctional equivalent is a "mimetic" compound, an organic chemicalconstruct designed to mimic the proper configuration and/or orientationfor targeting moiety-target cell binding. Another targeting moietyfunctional equivalent is a short polypeptide designated as a "minimal"polypeptide, constructed using computer-assisted molecular modeling andmutants having altered binding affinity, which minimal polypeptidesexhibit the binding affinity of the targeting moiety.

Preferred targeting moieties of the present invention are antibodies(polyclonal or monoclonal), peptides, oligonucleotides or the like.Polyclonal antibodies useful in the practice of the present inventionare polyclonal (Vial and Callahan, Univ. Mich. Med. Bull., 20: 284-6,1956), affinity-purified polyclonal or fragments thereof (Chao et al.,Res. Comm. in Chem. Path. & Pharm., 9: 749-61, 1974).

Monoclonal antibodies useful in the practice of the present inventioninclude whole antibody and fragments thereof. Such monoclonal antibodiesand fragments are producible in accordance with conventional techniques,such as hybridoma synthesis, recombinant DNA techniques and proteinsynthesis. Useful monoclonal antibodies and fragments may be derivedfrom any species (including humans) or may be formed as chimericproteins which employ sequences from more than one species. See,generally, Kohler and Milstein, Nature, 256: 495-97, 1975; Eur. J.Immunol., 6: 511-19, 1976.

Human monoclonal antibodies or "humanized" murine antibody are alsouseful as targeting moieties in accordance with the present invention.For example, murine monoclonal antibody may be "humanized" bygenetically recombining the nucleotide sequence encoding the murine Fvregion (i.e., containing the antigen binding sites) or thecomplementarity determining regions thereof with the nucleotide sequenceencoding a human constant domain region and an Fc region, e.g., in amanner similar to that disclosed in European Patent Application No.0,411,893 A2. Some murine residues may also be retained within the humanvariable region framework domains to ensure proper target site bindingcharacteristics. Humanized targeting moieties are recognized to decreasethe immunoreactivity of the antibody or polypeptide in the hostrecipient, permitting an increase in the half-life and a reduction inthe possibility of adverse immune reactions.

Types of active agents (diagnostic or therapeutic) useful herein includetoxins, anti-tumor agents, drugs and radionuclides. Several of thepotent toxins useful within the present invention consist of an A and aB chain. The A chain is the cytotoxic portion and the B chain is thereceptor-binding portion of the intact toxin molecule (holotoxin).Because toxin B chain may mediate non-target cell binding, it is oftenadvantageous to conjugate only the toxin A chain to a targeting protein.However, while elimination of the toxin B chain decreases non-specificcytotoxicity, it also generally leads to decreased potency of the toxinA chain-targeting protein conjugate, as compared to the correspondingholotoxin-targeting protein conjugate.

Preferred toxins in this regard include holotoxins, such as abrin,ricin, modeccin, Pseudomonas exotoxin A, Diphtheria toxin, pertussistoxin and Shiga toxin; and A chain or "A chain-like" molecules, such asricin A chain, abrin A chain, modeccin A chain, the enzymatic portion ofPseudomonas exotoxin A, Diphtheria toxin A chain, the enzymatic portionof pertussis toxin, the enzymatic portion of Shiga toxin, gelonin,pokeweed antiviral protein, saporin, tritin, barley toxin and snakevenom peptides. Ribosomal inactivating proteins (RIPs), naturallyoccurring protein synthesis inhibitors that lack translocating andcell-binding ability, are also suitable for use herein.

Extremely highly toxic toxins, such as palytoxin, blocked ricin (seeU.S. Pat. No. 5,239,062), bacterial toxins such as pseudomonas exotoxin,diphtheria toxin, fungal secondary metabolite toxins such astrichothecenes (e.g., roridin A and verrucarin A) and other highly toxicagents, such as potent chemotherapeutic agents such as actinomycin D andthe like, are also contemplated for use in the practice of the presentinvention. Such extremely highly toxic molecules exhibit toxicities atmicromolar and picomolar concentrations.

For example, palytoxin molecules having a preserved free terminal aminogroup are approximately 500-fold more toxic than palytoxin derivativeslacking such a free amine. In monkeys, palytoxin was found to have aLD50 of approximately 80 ng/kg. Consequently, an amino-derivatizedpalytoxin molecule is expected to exhibit an i.v. LD50 of approximately40 μg/kg. Palytoxin is set forth herein as a prototypical moleculecharacterized by extremely high toxicity. Palytoxin is also set forthherein as a prototype of a membrane active drug. Other examples ofmembrane active drugs are amphotericin B, polymyxin B and the like.

In conventional targeted therapy, an active agent is bound to anantibody or other targeting moiety to form the diagnostic or therapeuticconjugate to be administered. The accretion of active agent to targetsites is therefore dictated by the pharmacokinetics of the targetingmoiety. Whole monoclonal antibodies, for example, generally requireabout 20-72 hours to achieve optimal target site accretion, whileantibody fragments such as Fab and Fab' fragments generally requireabout 0-8 hours and F(ab').sub. 2 fragments generally require about 8-24hours. Consequently, the conjugate recipient's normal tissues areexposed to the active agent for the accretion time, leading toundesirable normal tissue toxicity. As a result of this normal tissueexposure, extremely highly toxic moieties cannot generally be employedin targeted therapy.

In the pretargeting approach; however, the pharmacokinetics of theactive agent is decoupled from that of the targeting moiety. Thetargeting moiety is permitted to accrete to target sites whileconjugated to a member of a ligand-anti-ligand pair. After thataccretion occurs and substantially all of the non-targeted conjugate iscleared from the recipient's circulation, the highly toxic active agentis administered as a conjugate to the complementary member of theligand-anti-ligand pair. Preferably, the toxin-ligand ortoxin-anti-ligand has a short serum half life and is excreted via therenal pathway. In this manner, the toxic active agent either accretes tothe target site where exertion of its toxic capability is desired, or itis rapidly removed from the recipient. This biodistribution of activeagent facilitates the protection of normal tissues of the recipient fromundesired toxicity. To enhance renal excretion, conjugation to a renalexcretion promoting biodistribution directing molecule as discussedbelow with regard to trichothecenes may be employed. Alternatively,lower, therapeutically effective doses of active agent may be employed.

Palytoxin, the prototypical extremely highly toxic active agent, ischaracterized by the following: non-proteinaceous structure having a2681 dalton molecular weight which exerts activity extracellularly bybinding to cell surface receptor and creating a pore in cell membranes.The palytoxin structure is known and is described in Bignami et al.,Cancer Research, 52:5759-5764, 1992.

Palytoxin exhibits the following functional characteristics:cytotoxicity against cultures of lymphocytes, fibroblasts and normal orvirus transformed epithelial cells. Palytoxin also depolarizes and lysesmammalian erythrocytes. Palytoxin appears to kill cells that express theNa⁺ K⁺ -ATPase-associated toxin receptor, in contrast anticancer agentswhich are selectively toxic to metabolically active cycling cells.Pharmacological studies indicate that palytoxin greatly perturbs thesodium, potassium and calcium fluxes in cells. This perturbation causesa cascade of events, including damage to mitochondria as well as releaseof protease and phospholipase enzymes, and, ultimately, results indamage to the ultrastructure of the cell membrane.

For use in pretargeting methods of the present invention, palytoxin isconjugated to a ligand or to an anti-ligand. For illustrative purposes,palytoxin-biotin and palytoxin-streptavidin conjugation are discussedbelow and in Example XVI.

One general strategy for preparing a biotin-palytoxin conjugate is asfollows:

1) protect the terminal amine nitrogen of palytoxin; and

2) alkylate a palytoxin hydroxyl group with a biotin-bearing alkylatingagent such as an iodoacetyl or a bromoacetyl biotin derivative. Thisstrategy does not afford selective functionalization of palytoxin,however. That is, the specific palytoxin hydroxyl group(s) thatparticipate in the reaction are not preselected.

Iodoacetyl-long chain (LC) biotin (available from Pierce ChemicalCompany) may be reacted with amine-protected palytoxin in DMF and NaH toform a biotin-palytoxin conjugate. The degree of biotin derivatizationof palytoxin will depend, among other things, upon the offering ratio ofiodoacetyl-LC-biotin:palytoxin. Generally, biotin derivatization rangingfrom 1 to about 5 biotins/palytoxin will be employed. Deprotection ofthe terminal palytoxin amine group may be conducted prior toadministration to a recipient of the palytoxin-biotin conjugate. Inthese circumstances, it is preferred that an acid or base cleavableprotecting group, such as BOC, TFA or the like, is used in theprotection step. If in vivo deprotection is contemplated, functionalgroups that are susceptible to enzyme cleavage (such as those discussedbelow with regard to cleavable linkages) are preferred. Similar routesmay be employed using alternative iodoacetyl-linker-biotin molecules.

Alternatively, a ligand or an anti-ligand may be linked to palytoxinselectively. That is, the linkage may be formed employing a uniquefunctionality of the palytoxin molecule. For example, a COOH moiety maybe liberated by treating a protected amine palytoxin derivative withpeptidase. An active ester is then formed using that liberated COOH, andthe active ester so formed is reacted with a derivatized biotin aminogroup. Still another possibility is derivatization at the C-55 position(unique acetal functionality) of the palytoxin structure. Followingprotection of the free palytoxin amine, the latent ketone in the ringstructure may be derivatized with a biotin amine in the presence ofNaCNBH₃. The product of the reductive animation may be represented asfollows:

Biotin-Linker-NH-CH₂ -palytoxin

where the carbon atom is the C-55 atom in the palytoxin structure andthe Linker is the non-amine portion of a homo- or hetero-bifunctionallinker bearing at least one amine group.

Another general palytoxin-biotin conjugate useful in the practice of thepresent invention is coupled via the terminal amine of the palytoxinmolecule. For example, an active ester biotin derivative such asN-hydroxysuccinimido-biotin, or a cleavable linker, may be coupled withpalytoxin bearing a free, unprotected terminal amine group at pH 7.5.

Preferred cleavable linkers useful in the practice of the presentinvention are linkers characterized by a physiological half-life greaterthan the time necessary for accretion of the palytoxin-biotin conjugateto target sites. Preferably, the physiological stability half-life ofthe linker is from about 2 to about 5 times the serum clearance of thebiotinylated palytoxin conjugate. In this manner, streptavidin ispretargeted to target sites and substantially removed from therecipient's circulation; biotin-cleavable linker-palytoxin isadministered and localizes to the pretargeted streptavidin; andpalytoxin is released from the conjugate and binds to a target sitereceptor. Consequently, the palytoxin delivered to the target site willbe in its native, more highly toxic, free amine form. Exemplarycleavable linkers useful in the practice of the present invention arehydrazido thiourea linkers (formed, for example, by reaction of NH₂-derivatized palytoxin isothiocyanate and biotin hydrazide); long chainamide linkers susceptible to biotinidase (formed, for example, from freeamine-bearing palytoxin and long chain-biotin-NHS ester available fromSigma Chemical Co., St. Louis, Mo.); ester linkers susceptible toesterases (formed, for example, from free-amine bearing palytoxin and anactive ester form of the reaction product of biotin andHO--CO--(CH₂)_(n) --Br where n ranges from 2 to 5); or the like.

Palytoxin-streptavidin conjugates may be employed, for example, intwo-step pretargeting protocols of the present invention. That is, abiotinylated targeting moiety may be administered and permitted tolocalize to target. Optionally, circulating biotin-targeting moietyconjugate may be cleared using a clearing agent (e.g.,galactosylated-avidin or the like) or other clearance mechanism. Next,palytoxin-streptavidin conjugate is administered and binds to thepretargeted biotin-containing conjugate. The streptavidin component ofthe conjugate may also decrease the whole body toxicity of palytoxin bybinding the free amine group of the palytoxin or by the folding ofstreptavidin relative to the palytoxin structure. Palytoxin-streptavidinconjugation may be conducted employing the COOH-liberated palytoxinderivative described above, for example.

Alternatively, a prodrug (i.e., inactive) form of palytoxin, such asN-(4'-hydroxyphenylacetyl)-palytoxin described in Bignami et al., CancerResearch, 52: 5759-5764, 1992, and activated by penicillin G amidase(PGA), may be employed in two-step or three-step pretargeting protocolsof the present invention.

A two-step pretargeting embodiment of the present invention involves:

administering to the recipient a first conjugate comprising a targetingmoiety; a member of a ligand-anti-ligand binding pair; and an enzymecapable of activating a prodrug, wherein the first conjugate localizesto a target site;

optionally administering to the recipient a clearing agent capable ofdirecting the clearance of circulating conjugate from the recipient oroptionally treating the recipient with a clearing device or analternative clearing procedure to substantially remove circulatingconjugate from the recipient; and

administering to the recipient a second conjugate comprising a prodrugand a ligand/anti-ligand binding pair member, wherein the secondconjugate binding pair member is complementary to that of the firstconjugate and wherein the second conjugate is cleared from the recipientrapidly and, preferably, via the renal pathway.

For example, an non-internalizing, anti-carcinoma IgG_(2a) antibody,such as L6, may be conjugated to streptavidin and PGA via techniquesdescribed herein as well as art-recognized methods therefor. ThisPGA-L6-streptavidin conjugate is administered and permitted to localizeto target sites. Preferably, a clearing agent, such asgalactose-HSA-biotin is administered at a later time point to facilitateclearance of circulating PGA-L6-streptavidin. A few hours later,N-(4'-hydroxyphenylacetyl)-palytoxin-biotin conjugate is administeredand either accretes to pretargeted PGA-L6-streptavidin or is eliminatedfrom the recipient's circulation by the recipient's endogenousmechanisms therefor.

In alternative two-step protocols, the ligand/anti-ligand interaction isone involving enzymes and enzyme inhibitors. Such a two-steppretargeting protocol includes:

administering to the recipient a first conjugate comprising a targetingmoiety; and an enzyme capable of activating a prodrug, wherein the firstconjugate localizes to a target site;

optionally administering to the recipient a clearing agent comprising anenzyme inhibitor capable of directing the clearance of circulatingconjugate from the recipient or optionally treating the recipient with aclearing device or an alternative clearing procedure to substantiallyremove circulating conjugate from the recipient; and

administering to the recipient a prodrug, wherein the prodrug isconverted into active, cytotoxic form at the sites of pretargetedenzyme.

For example, an L6-PGA conjugate may be administered and permitted tolocalize to target sites. Preferably, a clearing agent, such asgalactose-HSA-PGA irreversible inhibitor or a conjugate incorporating areversible or irreversible PGA inhibitor and a large, non-extravascularpermeating molecule is administered at a later time point to facilitateclearance of circulating PGA-L6-streptavidin.

An exemplary large, non-extravascular permeating molecule, is dextran.Other polymers, polymeric particulates or liposomes, as discussedelsewhere herein, may also be employed.

Exemplary irreversible inhibitors useful in the practice of the presentinvention may be designed by incorporating a reactive group, such as aniodoacetyl group or an amide group, in a molecule that resembles asubstrate. Iodoacetamide, for example, is an irreversible inhibitor ofmany enzymes that contain a cysteine residue in the active site.Exemplary reversible inhibitors of PGA are other amide substrates, e.g.,peptides such as triglycine or 4-hydroxyphenylacetylglycine and thelike.

A few hours later, a prodrug form of an active agent is administered.For example, N-(4'-hydroxyphenyl-acetyl)-palytoxin is administered,which molecule either accretes to pretargeted PGA-L6 or is eliminatedfrom the recipient's circulation by the recipient's endogenousmechanisms therefor.

One alternative to the optional clearance step set forth above is simplyto allow an amount of time to pass that is sufficient to permit therecipient's native clearance mechanisms to substantially removecirculating first conjugate.

The three-step approach involves:

administering to the recipient a first conjugate comprising a targetingmoiety; an enzyme; and a ligand, wherein the first conjugate localizesto a target site;

administering to the recipient an anti-ligand; or aanti-ligand-containing conjugate; and

administering to the recipient a prodrug, wherein the prodrug isconverted to active, cytotoxic form at sites of pretargeted enzyme.

While the methods set forth above have been described with regard to theN-(4'-hydroxyphenyl-acetyl)palytoxin/PGA prodrug/enzyme pair, thosemethods are amenable to other such pairs. An example of a prodrug/enzymepair useful in the practice of the present invention is a phosphate formof a drug (e.g., phenol mustard phosphate, etoposide phosphate,mitomycin phosphate, doxorubicin phosphate and the like) and an alkalinephosphatase enzyme. See, for example, Wallace et al., Bioconj. Chem., 2:349-352, 1991. Another example is 5-fluorocytosine (5FC)/cytosinedeaminase (CDase), described in Senter et al., Bioconj. Chem., 2:447-451, 1991. An additional example involves activation of beta-lactamprodrugs by beta-lactamase. See, for example, Meyer et al., Bioconj.Chem., 3: 42-48, 1992.

Preferred drugs suitable for use herein include conventionalchemotherapeutics, such as vinblastine, doxorubicin, bleomycin,methotrexate, 5-fluorouracil, 6-thioguanine, cytarabine,cyclophosphamide and cis-platinum, as well as other conventionalchemotherapeutics as described in Cancer: Principles and Practice ofOncology, 2d ed., V. T. DeVita, Jr., S. Hellman, S. A. Rosenberg, J. B.Lippincott Co., Philadelphia, Pa., 1985, Chapter 14. A particularlypreferred drug within the present invention is a trichothecene.

Trichothecenes are drugs produced by soil fungi of the class Fungiimperfecti or isolated from Baccharus megapotamica (Bamburg, J. R. Proc.Molec. Subcell. Biol. 8:41-110, 1983; Jarvis & Mazzola, Acc. Chem. Res.15:338-395, 1982). They appear to be the most toxic molecules thatcontain only carbon, hydrogen and oxygen (Tamm, C. Fortschr. Chem. Org.Naturst. 31:61-117, 1974). They are all reported to act at the level ofthe ribosome as inhibitors of protein synthesis at the initiation,elongation, or termination phases.

There are two broad classes of trichothecenes: those that have only acentral sesquiterpenoid structure and those that have an additionalmacrocyclic ring (simple and macrocyclic trichothecenes, respectively).The simple trichothecenes may be subdivided into three groups (i.e.,Group A, B, and C) as described in U.S. Pat. Nos. 4,744,981 and4,906,452 (incorporated herein by reference). Representative examples ofGroup A simple trichothecenes include: Scirpene, Roridin C,dihydrotrichothecene, Scirpen-4, 8-diol, Verrucarol, Scirpentriol, T-2tetraol, pentahydroxyscirpene, 4-deacetylneosolaniol, trichodermin,deacetylcalonectrin, calonectrin, diacetylverrucarol,4-monoacetoxyscirpenol, 4,15-diacetoxyscirpenol,7-hydroxydiacetoxyscirpenol, 8-hydroxydiacetoxy-scirpenol (Neosolaniol),7,8-dihydroxydiacetoxyscirpenol, 7-hydroxy-8-acetyldiacetoxyscirpenol,8-acetylneosolaniol, NT-1, NT-2, HT-2, T-2, and acetyl T-2 toxin.Representative examples of Group B simple trichothecenes include:Trichothecolone, Trichothecin, deoxynivalenol, 3-acetyldeoxynivalenol,5-acetyldeoxynivalenol, 3,15-diacetyldeoxynivalenol, Nivalenol,4-acetylnivalenol (Fusarenon-X), 4,15-idacetylnivalenol,4,7,15-triacetylnivalenol, and tetra-acetylnivalenol. Representativeexamples of Group C simple trichothecenes include: Crotocol andCrotocin. Representative macrocyclic trichothecenes include VerrucarinA, Verrucarin B, Verrucarin J (Satratoxin C), Roridin A, Roridin D,Roridin E (Satratoxin D), Roridin H, Satratoxin F, Satratoxin G,Satratoxin H, Vertisporin, Mytoxin A, Mytoxin C, Mytoxin B, Myrotoxin A,Myrotoxin B, Myrotoxin C, Myrotoxin D, Roritoxin A, Roritoxin B, andRoritoxin D. In addition, the general "trichothecene" sesquiterpenoidring structure is also present in compounds termed "baccharins" isolatedfrom the higher plant Baccharis megapotamica, and these are described inthe literature, for instance as disclosed by Jarvis et al. (Chemistry ofAlleopathy, ACS Symposium Series No. 268: ed. A. C. Thompson, 1984, pp.149-159).

The present invention provides for effective delivery of active agentsincluding toxins. The decoupling of the pharmacokinetics of thetargeting moiety (generally slow), and the toxin (generally fast whenadministered intravenously, half-life less than about 5 minutes withlonger lived metabolites) in combination with the high affinityinteraction between a ligand-anti-ligand pair is responsible for thisimprovement. When the toxin active agents are not themselves generallyrapidly cleared (typically via the hepatic pathway), conjugatescontaining such active agents are preferably constructed to impartrelatively rapid, preferably renal, clearance thereto. Derivatization ofthe toxin with ligand or with anti-ligand may be insufficient toredirect the biodistribution of the toxin active agent. Alternatively,the active agent may be administered in a lower, but therapeuticallyeffective, dose. In this manner, the non-target tissue of the recipientof the toxin active agent does not suffer prolonged exposure to thetoxic active agent.

Toxin molecules, such as pseudomonas exotoxin (PE) and trichothecenes,are primarily metabolized in the liver. Consequently, liver toxicity isassociated with administration of PE. Also, administration of anti-tumoragents, such as IL-2 and TNF, has been shown to result in livertoxicity. Active agents characterized by such a biodistribution patternmay be accommodated in two-step or three-step pretargeting protocols ofthe present invention in two ways.

First, low doses of the active agent-ligand or active agent-anti-ligandmay be given. Because of the high affinity of ligand for thecomplementary anti-ligand, a therapeutically effective dose may bedelivered to the tumor, without the necessity for active agent-targetingmoiety binding during targeting moiety accretion to target site. Theactive agent-ligand or active agent-anti-ligand are generally processedby the recipient's liver. Because of the lower dose of active agentadministered to the recipient and the decreased circulation time of thatactive agent in the recipient (active agent circulation half-life isgenerally less than the time for maximum target site accretion of thetargeting moiety a therapeutically effective dose may be delivered tothe target site without an unmanageable level of toxicity beingdelivered to non-target sites. Doses of active agent, ranging fromnanograms to about micrograms may be administered in this manner, withthe attending physician being responsible for the dosing choice in lightof the condition and treatment history of the particular recipient.

Alternatively, the active agent may be coupled to a polymeric moleculeof sufficient size to direct the biodistribution of the conjugate to thekidneys. Suitable polymeric molecules preferably range in molecularweight between from about 5000 to about 50,000 daltons. Polymers of lessthan about 5000 daltons will not generally direct the biodistribution oflarge active agents. Polymers of greater than 50,000 daltons are likelyto be metabolized in the liver. Exemplary polymers useful in this aspectof the present invention include dextran, polylysine, polyglytamate,oligosaccharides of defined size (e.g., from about 5 to about 50 kD) andthe like. Methods for coupling dextran, for example, to ligands andanti-ligands are discussed in the examples below.

Active agent-polymer-ligand or -anti-ligand conjugates can beadministered at high active agent doses, because such conjugates exhibitrapid renal clearance. Consequently, non-target tissues of the recipientare exposed to the active agent for only a short time until the activeagent is either bound at the target site or processed via renalexcretion. As a result, doses of active agent, ranging from aboutmicrograms to about milligrams (10⁻⁶ to 10⁻³ M), may be administered inthis manner.

Experimental drugs, such as mercaptopurine, N-methylformamide,2-amino-1,3,4-thiadiazole, melphalan, hexamethylmelamine, galliumnitrate, 3% thymidine, dichloromethotrexate, mitoguazone, suramin,bromodeoxyuridine, iododeoxyuridine, semustine,1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea,N,N'-hexamethylene-bis-acetamide, azacitidine, dibromodulcitol, Erwiniaasparaginase, ifosfamide, 2-mercaptoethane sulfonate, teniposide, taxol,3-deazauridine, soluble Baker's antifol, homoharringtonine,cyclocytidine, acivicin, ICRF-187, spiromustine, levamisole,chlorozotocin, aziridinyl benzoquinone, spirogermanium, aclarubicin,pentostatin, PALA, carboplatin, amsacrine, caracemide, iproplatin,misonidazole, dihydro-5-azacytidine, 4'-deoxy-doxorubicin, menogaril,triciribine phosphate, fazarabine, tiazofurin, teroxirone, ethiofos,N-(2-hydroxyethyl)-2-nitro-1H-imidazole-1-acetamide, mitoxantrone,acodazole, amonafide, fludarabine phosphate, pibenzimol, didemnin B,merbarone, dihydrolenperone, flavone-8-acetic acid, oxantrazole,ipomeanol, trimetrexate, deoxyspergualin, echinomycin, anddideoxycytidine (see NCI Investigational Drugs, Pharmaceutical Data1987, NIH Publication No. 88-2141, Revised November 1987) are alsopreferred.

Radionuclides useful within the present invention includegamma-emitters, positron-emitters, Auger electron-emitters, X-rayemitters and fluorescence-emitters, with beta- or alpha-emitterspreferred for therapeutic use. Radionuclides are well-known in the artand include ¹²³ I, ¹²⁵ I, ¹³⁰ I, ¹³¹ I, ¹³³ I, ¹³⁵ I, ⁴⁷ Sc, ⁷² As, ⁷²Se, ⁹⁰ Y, ⁸⁸ Y, ⁹⁷ Ru, ¹⁰⁰ Pd, ¹⁰¹ Rh, ¹¹⁹ Sb, ¹²⁸ Ba, ¹⁹⁷ Hg, ²¹¹ At,²¹² Bi, ¹⁵³ Sm, ¹⁶⁹ Eu, ²¹² Pb, ¹⁰⁹ Pd, ¹¹¹ In, ⁶⁷ Ga, ⁶⁸ Ga, ⁶⁴ Cu, ⁶⁷Cu, ⁷⁵ Br, ⁷⁶ Br, ⁷⁷ Br, ^(99m) Tc, ¹¹ C, ¹³ N, ¹⁵ O, ¹⁶⁶ Ho and ¹⁸ F.Preferred therapeutic radionuclides include ¹⁸⁸ Re, ¹⁸⁶ Re, ²⁰³ Pb, ²¹²Pb, ²¹² Bi, ¹⁰⁹ Pd, ⁶⁴ Cu, ⁶⁷ Cu, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, ⁷⁷ Br, ²¹¹ At, ⁹⁷Ru, ¹⁰⁵ Rh, ¹⁹⁸ Au, ¹⁶⁶ Ho and ¹⁹⁹ Ag or ¹⁷⁷ Lu.

Other anti-tumor agents, e.g., agents active against proliferatingcells, are administrable in accordance with the present invention.Exemplary anti-tumor agents include cytokines and other moieties, suchas interleukins (e.g.,IL-2, IL-4, IL-6, IL-12 and the like),transforming growth factor-beta, lymphotoxin, tumor necrosis factor,interferons (e.g., gamma-interferon), colony stimulating factors (e.g.,GM-CSF, M-CSF and the like), vascular permeability factor or the like,lectin inflammatory response promoters (selectins), such as L-selectin,E-selectin, P-selectin or the like, proteinaceous moieties such as Clqand NK receptor protein, and like molecules.

Ligands suitable for use within the present invention include biotin,haptens, lectins, epitopes, dsDNA fragments, enzyme inhibitors andanalogs and derivatives thereof. Useful complementary anti-ligandsinclude avidin (for biotin), carbohydrates (for lectins) and antibody,fragments or analogs thereof, including mimetics (for haptens andepitopes) and zinc finger proteins (for dsDNA fragments) and enzymes(for enzyme inhibitors). Preferred ligands and anti-ligands bind to eachother with an affinity of at least about k_(D) ≧10⁹ M.

One component to be administered in a preferred two-step pretargetingprotocol is a targeting moiety-anti-ligand or a targeting moiety-ligandconjugate. In three-step pretargeting, a preferred component foradministration is a targeting moiety-ligand conjugate.

A preferred targeting moiety useful in these embodiments of the presentinvention is a monoclonal antibody. Protein-protein conjugations aregenerally problematic due to the formation of undesirable byproducts,including high molecular weight and cross-linked species, however. Anon-covalent synthesis technique involving reaction of biotinylatedantibody with streptavidin has been reported to result in substantialbyproduct formation. Also, at least one of the four biotin binding siteson the streptavidin is used to link the antibody and streptavidin, whileanother such binding site may be sterically unavailable for biotinbinding due to the configuration of the streptavidin-antibody conjugate.

Thus, covalent streptavidin-antibody conjugation is preferred, but highmolecular weight byproducts are often obtained. The degree ofcrosslinking and aggregate formation is dependent upon several factors,including the level of protein derivitization using heterobifunctionalcrosslinking reagents. Sheldon et al., i Appl. Radiat. Isot. 43:1399-1402, 1992, discuss preparation of covalent thioether conjugates byreacting succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(SMCC)-derivatized antibody and iminothiolane-derivatized streptavidin.

Streptavidin-proteinaceous targeting moiety conjugates are preferablyprepared as described in Example XI below, with the preparationinvolving the steps of: preparation of SMCC-derivatized streptavidin;preparation of DTT-reduced proteinaceous targeting moiety; conjugationof the two prepared moieties; and purification of the monosubstituted ordisubstituted (with respect to streptavidin) conjugate from crosslinked(antibody-streptavidin-antibody) and aggregate species and unreactedstarting materials. The purified fraction is preferably furthercharacterized by one or more of the following techniques: HPLC sizeexclusion, SDS-PAGE, immunoreactivity, biotin binding capacity and invivo studies.

Alternatively, thioether conjugates useful in the practice of thepresent invention may be formed using other thiolating agents, such asSPDP, iminothiolane, SATA or the like, or other thio-reactiveheterobifunctional cross linkers, such asm-maleimidobenzoyl-N-hydroxysuccinimide ester,N-succinimidyl(4-iodoacetyl)aminobenzoate or the like.

Streptavidin-proteinaceous targeting moiety conjugates of the presentinvention can also be formed by conjugation of a lysine epsilon aminogroup of one protein with a maleimide-derivatized form of the otherprotein. For example, at pH 8-10, lysine epsilon amino moieties reactwith protein maleimides, prepared, for instance, by treatment of theprotein with SMCC, to generate stable amine covalent conjugates. Inaddition, conjugates can be prepared by reaction of lysine epsilon aminomoieties of one protein with aldehyde functionalities of the otherprotein. The resultant imine bond is reducible to generate thecorresponding stable amine bond. Aldehyde functionalities may begenerated, for example, by oxidation of protein sugar residues or byreaction with aldehyde-containing heterobifunctional cross linkers.

Another method of forming streptavidin-targeting moiety conjugatesinvolves immobilized iminobiotin that binds SMCC-derivatizedstreptavidin. In this conjugation/purification method, the reversiblebinding character of iminobiotin (immobilized) to streptavidin isexploited to readily separate conjugate from the unreacted targetingmoiety. Iminobiotin binding can be reversed under conditions of lower pHand elevated ionic strength, e.g., NH₂ OAc, pH 4 (50 mM) with 0.5 MNaCl.

For streptavidin, for example, the conjugation/purification proceeds asfollows:

SMCC-derivatized streptavidin is bound to immobilized iminobiotin(Pierce Chemical Co., St. Louis, Mo.), preferably in column format;

a molar excess (with respect to streptavidin) of DTT-reduced antibody(preferably free of reductant) is added to the nitrogen-purged,phosphate-buffered iminobiotin column wherein the SMCC-streptavidin isbound (DTT-reduced antibody will saturate the bound SMCC-streptavidin,and unbound reduced antibody passing through the column can be reused);

the column is washed free of excess antibody; and

a buffer that lowers the pH and increases ionic strength is added to thecolumn to elute streptavidin-antibody conjugate in pure form.

As indicated above, targeting moiety-mediated ligand-anti-ligandpretargeting involves the localization of either targeting moiety-ligandor targeting moiety-anti-ligand at target tissue. Often, peak uptake tosuch target tissue is achieved before the circulating level of targetingmoiety-containing conjugate in the blood is sufficiently low to permitthe attainment of an optimal target-to-non-target conjugate ratio. Toobviate this problem, two approaches are useful. The first approachallows the targeting moiety-containing conjugate to clear from the bloodby "natural" or endogenous clearance mechanisms. This method iscomplicated by variations in systemic clearance of proteins and byendogenous ligand or anti-ligand. For example, endogenous biotin mayinterfere with the preservation of biotin binding sites on astreptavidin-targeting moiety conjugate.

The second approach for improving targeting moiety-ligand or targetingmoiety-anti-ligand conjugate target-to-blood ratio "chases" theconjugate from the circulation through in vivo complexation of conjugatewith a molecule constituting or containing the complementary anti-ligandor ligand. When biotinylated antibodies are used as a ligand-targetingmoiety conjugate, for example, avidin forms relatively large aggregatedspecies upon complexation with the circulating biotinylated antibody,which aggregated species are rapidly cleared from the blood by the RESuptake. See, for example, U.S. Pat. No. 4,863,713. One problem with thismethod, however, is the potential for cross-linking and internalizingtumor-bound biotinylated antibody by avidin.

When avidin-targeting moiety conjugates are employed, poly-biotinylatedtransferrin has been used to form relatively large aggregated speciesthat are cleared by RES uptake. See, for example, Goodwin, J. Nucl. Med.33(10):1816-18, 1992). Poly-biotinylated transferrin also has thepotential for cross-linking and internalizing tumor-boundavidinylated-targeting moiety, however. In addition, both "chase"methodologies involve the prolonged presence of aggregated moieties ofintermediate, rather than large, size (which are not cleared as quicklyas large size particles by RES uptake), thereby resulting in serumretention of subsequently administered ligand-active agent oranti-ligand-active agent. Such serum retention unfavorably impacts thetarget cell-to-blood targeting ratio.

The present invention provides clearing agents of protein andnon-protein composition having physical properties facilitating use forin vivo complexation and blood clearance of anti-ligand/ligand (e.g.,avidin/biotin)-targeting moiety (e.g., antibody) conjugates. Theseclearing agents are useful in improving the target:blood ratio oftargeting moiety conjugate. Other applications of these clearing agentsinclude lesional imaging or therapy involving blood clots and the like,employing antibody-active agent delivery modalities. For example,efficacious anti-clotting agent provides rapid target localization andhigh target:non-target targeting ratio. Active agents administered inpretargeting protocols of the present invention using efficient clearingagents are targeted in the desirable manner and are, therefore, usefulin the imaging/therapy of conditions such as pulmonary embolism and deepvein thrombosis.

Clearing agents useful in the practice of the present inventionpreferably exhibit one or more of the following characteristics:

rapid, efficient complexation with targeting moiety-ligand (oranti-ligand) conjugate in vivo;

rapid clearance from the blood of targeting moiety conjugate capable ofbinding a subsequently administered complementary anti-ligand or ligandcontaining molecule;

high capacity for clearing (or inactivating) large amounts of targetingmoiety conjugate; and

low immunogenicity.

Preferred clearing agents include hexose-based and non-hexose basedmoieties. Hexose-based clearing agents are molecules that have beenderivatized to incorporate one or more hexoses (six carbon sugarmoieties) recognized by Ashwell receptors or other receptors such as themannose/N-acetylglucosamine receptor which are associated withendothelial cells and/or Kupffer cells of the liver or the mannose6-phosphate receptor. Exemplary of such hexoses are galactose, mannose,mannose 6-phosphate, N-acetylglucosamine and the like. Other moietiesrecognized by Ashwell receptors, including glucose, N-galactosamine,N-acetylgalactosamine, thioglycosides of galactose and, generally,D-galactosides and glucosides or the like may also be used in thepractice of the present invention. Galactose is the prototypicalclearing agent hexose derivative for the purposes of this description.Galactose thioglycoside conjugation to a protein is preferablyaccomplished in accordance with the teachings of Lee et al.,"2-Imino-2-methoxyethyl 1-Thioglycosides: New Reagents for AttachingSugars to Proteins," Biochemistry, 15)(18): 3956, 1976. Another usefulgalactose thioglycoside conjugation method is set forth in Drantz et al,"Attachment of Thioglycosides to Proteins: Enhancement of Liver MembraneBinding," Biochemistry, 15)(18): 3963, 1976. Thus, galactose-based andnon-galactose based molecules are discussed below.

Protein-type galactose-based clearing agents include proteins havingendogenous exposed galactose residues or which have been derivatized toexpose or incorporate such galactose residues. Exposed galactoseresidues direct the clearing agent to rapid clearance by endocytosisinto the liver through specific receptors therefor (Ashwell receptors).These receptors bind the clearing agent, and induce endocytosis into thehepatocyte, leading to fusion with a lysosome and recycle of thereceptor back to the cell surface. This clearance mechanism ischaracterized by high efficiency, high capacity and rapid kinetics.

An exemplary clearing agent of the protein-based/galactose-bearingvariety is the asialoorosomucoid derivative of human alpha-1 acidglycoprotein (orosomucoid, molecular weight=41,000 Dal, isoelectricpoint=1.8-2.7). The rapid clearance from the blood of asialoorosomucoidhas been documented by Galli, et al., J. of Nucl. Med. Allied Sci.32)(2): 110-16, 1988.

Treatment of orosomucoid with neuraminidase removes sialic acidresidues, thereby exposing galactose residues. Other such derivatizedclearing agents include, for example, galactosylated albumin,galactosylated-IgM, galactosylated-IgG, asialohaptoglobin, asialofetuin,asialoceruloplasmin and the like.

Human serum albumin (HSA), for example, may be employed in a clearingagent of the present invention as follows:

(Hexose)_(m) --Human Serum Albumin (HSA)--(Ligand)_(n), wherein n is aninteger from 1 to about 10 and m is an integer from 1 to about 25 andwherein the hexose is recognized by Ashwell receptors. In a preferredembodiment of the present invention the ligand is biotin and the hexoseis galactose. More preferably, HSA is derivatized with from 10-20galactose residues and 1-5 biotin residues. Still more preferably, HSAclearing agents of the present invention are derivatized with from about12 to about 15 galactoses and 3 biotins. Derivatization with bothgalactose and biotin are conducted in a manner sufficient to produceindividual clearing agent molecules with a range of biotinylation levelsthat averages a recited whole number, such as 1, biotin. Derivatizationwith 3 biotins, for example, produces a product mixture made up ofindividual clearing agent molecules, substantially all of which havingat least one biotin residue. Derivatization with 1 biotin produces aclearing agent product mixture, wherein a significant portion of theindividual molecules are not biotin derivatized. The whole numbers usedin this description refer to the average biotinylation of the clearingagents under discussion.

In addition, clearing agents based upon human proteins, especially humanserum proteins, such as, for example, orosomucoid and human serumalbumin, human IgG, human-anti-antibodies of IgG and IgM class and thelike, are less immunogenic upon administration into the serum of a humanrecipient. Another advantage of using asialoorosomucoid is that humanorosomucoid is commercially available from, for example, Sigma ChemicalCo, St. Louis, Mo.

One way to prevent clearing agent compromise of target-bound conjugatethrough direct complexation is through use of a clearing agent of a sizesufficient to render the clearing agent less capable of diffusion intothe extravascular space and binding to target-associated conjugate. Thisstrategy is useful alone or in combination with the aforementionedrecognition that exposed galactose residues direct rapid liver uptake.This size-exclusion strategy enhances the effectiveness ofnon-galactose-based clearing agents of the present invention. Thecombination (exposed galactose and size) strategy improves theeffectiveness of "protein-type" or "polymer-type" galactose-basedclearing agents.

Galactose-based clearing agents include galactosylated, biotinylatedproteins (to remove circulating streptavidin-targeting moietyconjugates, for example) of intermediate molecular weight (ranging fromabout 40,000 to about 200,000 Dal), such as biotinylatedasialoorosomucoid, galactosyl-biotinyl-human serum albumin or othergalactosylated and biotinylated derivatives of non-immunogenic solublenatural proteins, as well as biotin- and galactose-derivatizedpolyglutamate, polylysine, polyarginine, polyaspartate and the like.High molecular weight moieties (ranging from about 200,000 to about1,000,000 Dal) characterized by poor target access, includinggalactosyl-biotinyl-IgM or -IgG (approximately 150,000 Dal) molecules,as well as galactose- and biotin-derivatized transferrin conjugates ofhuman serum albumin, IgG and IgM molecules and the like, can also beused as clearing agents of the claimed invention. Chemically modifiedpolymers of intermediate or high molecular weight (ranging from about40,000 to about 1,000,000 Dal), such as galactose- andbiotin-derivatized dextran, hydroxypropylmethacrylamide polymers,polyvinylpyrrolidone-polystyrene copolymers, divinyl ether-maleic acidcopolymers, pyran copolymers, or PEG, also have utility as clearingagents in the practice of the present invention. In addition, rapidlyclearing biotinylated liposomes (high molecular weight moieties withpoor target access) can be derivatized with galactose and biotin toproduce clearing agents for use in the practice of the presentinvention.

A further class of clearing agents useful in the present inventioninvolve small molecules (ranging from about 500 to about 10,000 Dal)derivatized with galactose and biotin that are sufficiently polar to beconfined to the vascular space as an in vivo volume of distribution.More specifically, these agents exhibit a highly charged structure and,as a result, are not readily distributed into the extravascular volume,because they do not readily diffuse across the lipid membranes liningthe vasculature. Exemplary of such clearing agents are mono- orpoly-biotin-derivatized6,6'-[(3,3'-dimethyl[1,1'-biphenyl]-4,4'-diyl)bis(azo)bis[4-amino-5-hydroxy-1,3-naphthalene disulfonic acid] tetrasodium salt,mono- or poly-biotinyl-galactose-derivatized polysulfateddextran-biotin, mono- or poly-biotinyl-galactose-derivatizeddextran-biotin and the like.

The galactose-exposed or -derivatized clearing agents are preferablycapable of (1) rapidly and efficiently complexing with the relevantligand- or anti-ligand-containing conjugates via ligand-anti-ligandaffinity; and (2) clearing such complexes from the blood via thegalactose receptor, a liver specific degradation system, as opposed toaggregating into complexes that are taken up by the generalized RESsystem, including the lung and spleen. Additionally, the rapid kineticsof galactose-mediated liver uptake, coupled with the affinity of theligand-anti-ligand interaction, allow the use of intermediate or evenlow molecular weight carriers.

Non-galactose residue-bearing moieties of low or intermediate molecularweight (ranging from about 40,000 to about 200,000 Dal) localized in theblood may equilibrate with the extravascular space and, therefore, binddirectly to target-associated conjugate, compromising targetlocalization. In addition, aggregation-mediated clearance mechanismsoperating through the RES system are accomplished using a largestoichiometric excess of clearing agent. In contrast, the rapid bloodclearance of galactose-based clearing agents used in the presentinvention prevents equilibration, and the high affinityligand-anti-ligand binding allows the use of low stoichiometric amountsof such galactose-based clearing agents. This feature further diminishesthe potential for galactose-based clearing agents to compromisetarget-associated conjugate, because the absolute amount of suchclearing agent administered is decreased.

Clearing agent evaluation experimentation involving galactose- andbiotin-derivatized clearing agents of the present invention is detailedin Examples XIII and XV. Specific clearing agents of the presentinvention that were examined during the Example XV experimentation are(1) asialoorosomucoid-biotin, (2) human serum albumin derivatized withgalactose and biotin, and (3) a 70,000 dalton molecular weight dextranderivatized with both biotin and galactose. The experimentation showedthat proteins and polymers are derivatizable to contain both galactoseand biotin and that the resultant derivatized molecule is effective inremoving circulating streptavidin-protein conjugate from the serum ofthe recipient. Biotin loading was varied to determine the effects onboth clearing the blood pool of circulating avidin-containing conjugateand the ability to deliver a subsequently administered biotinylatedisotope to a target site recognized by the streptavidin-containingconjugate. The effect of relative doses of the administered componentswith respect to clearing agent efficacy was also examined.

Protein-type and polymer-type non-galactose-based clearing agentsinclude the agents described above, absent galactose exposure orderivitization and the like. These clearing agents act through anaggregation-mediated RES mechanism. In these embodiments of the presentinvention, the clearing agent used will be selected on the basis of thetarget organ to which access of the clearing agent is to be excluded.For example, high molecular weight (ranging from about 200,000 to about1,000,000 Dal) clearing agents will be used when tumor targets or clottargets are involved.

Another class of clearing agents includes agents that do not removecirculating ligand or anti-ligand/targeting moiety conjugates, butinstead "inactivate" the circulating conjugates by blocking the relevantanti-ligand or ligand binding sites thereon. These "cap-type" clearingagents are preferably small (500 to 10,000 Dal) highly chargedmolecules, which exhibit physical characteristics that dictate a volumeof distribution equal to that of the plasma compartment (i.e., do notextravasate into the extravascular fluid volume). Exemplary cap-typeclearing agents are poly-biotin-derivatized6,6'-[(3,3'-dimethyl[1,1'-biphenyl]-4,4'-diyl)bis(azo)bis[4-amino-5-hydroxy-1,3-naphthalene disulfonic acid] tetrasodium salt,poly-biotinyl-derivatized polysulfated dextran-biotin, mono- orpoly-biotinyl-derivatized dextran-biotin and the like.

Cap-type clearing agents are derivatized with the relevant anti-ligandor ligand, and then administered to a recipient of previouslyadministered ligand/ or anti-ligand/targeting moiety conjugate. Clearingagent-conjugate binding therefore diminishes the ability of circulatingconjugate to bind any subsequently administered active agent-ligand oractive agent-anti-ligand conjugate. The ablation of active agent bindingcapacity of the circulating conjugate increases the efficiency of activeagent delivery to the target, and increases the ratio of target-boundactive agent to circulating active agent by preventing the coupling oflong-circulating serum protein kinetics with the active agent. Also,confinement of the clearing agent to the plasma compartment preventscompromise of target-associated ligand or anti-ligand.

Clearing agents of the present invention may be administered in singleor multiple doses. A single dose of biotinylated clearing agent, forexample, produces a rapid decrease in the level of circulating targetingmoiety-streptavidin, followed by a small increase in that level,presumably caused, at least in part, by re-equilibration of targetingmoiety-streptavidin within the recipient's physiological compartments. Asecond or additional clearing agent doses may then be employed toprovide supplemental clearance of targeting moiety-streptavidin.Alternatively, clearing agent may be infused intravenously for a timeperiod sufficient to clear targeting moiety-streptavidin in a continuousmanner.

Other types of clearing agents and clearance systems are also useful inthe practice of the present invention to remove circulating targetingmoiety-ligand or -anti-ligand conjugate from the recipient'scirculation. Particulate-based clearing agents, for example, arediscussed in Example IX. In addition, extracorporeal clearance systemsare discussed in Example IX. In vivo clearance protocols employingarterially inserted proteinaceous or polymeric multiloop devices arealso described in Example IX.

One embodiment of the present invention in which rapid acting clearingagents are useful is in the delivery of Auger emitters, such as I-125,I-123, Er-165, Sb-119, Hg-197, Ru-97, Tl-201 and I-125 and Br-77, ornucleus-binding drugs to target cell nuclei. In these embodiments of thepresent invention, targeting moieties that localize to internalizingreceptors on target cell surfaces are employed to deliver a targetingmoiety-containing conjugate (i.e., a targeting moiety-anti-ligandconjugate in the preferred two-step protocol) to the target cellpopulation. Such internalizing receptors include EGF receptors,transferrin receptors, HER2 receptors, IL-2 receptors, otherinterleukins and cluster differentiation receptors, somatostatinreceptors, other peptide binding receptors and the like.

After the passage of a time period sufficient to achieve localization ofthe conjugate to target cells, but insufficient to induceinternalization of such targeted conjugates by those cells through areceptor-mediated event, a rapidly acting clearing agent isadministered. In a preferred two-step protocol, an activeagent-containing ligand or anti-ligand conjugate, such as a biotin-Augeremitter or a biotin-nucleus acting drug, is administered as soon as theclearing agent has been given an opportunity to complex with circulatingtargeting moiety-containing conjugate, with the time lag betweenclearing agent and active agent administration being less than about 24hours. In this manner, active agent is readily internalized throughtarget cell receptor-mediated internalization. While circulating Augeremitters are thought to be non-toxic, the rapid, specific targetingafforded by the pretargeting protocols of the present inventionincreases the potential of shorter half-life Auger emitters, such asI-123, which is available and capable of stable binding.

In order to more effectively deliver a therapeutic or diagnostic dose ofradiation to a target site, the radionuclide is preferably retained atthe tumor cell surface. Loss of targeted radiation occurs as aconsequence of metabolic degradation mediated by metabolically activetarget cell types, such as tumor or liver cells.

Preferable agents and protocols within the present invention aretherefore characterized by prolonged residence of radionuclide at thetarget cell site to which the radionuclide has localized and improvedradiation absorbed dose deposition at that target cell site, withdecreased targeted radioactivity loss resulting from metabolism.Radionuclides that are particularly amenable to the practice of thisaspect of the present invention are rhenium, iodine and like "non +3charged" radiometals which exist in chemical forms that easily crosscell membranes and are not, therefore, inherently retained by cells. Incontrast, radionuclides having a +3 charge, such as In-111, Y-90, Lu-177and Ga-67, exhibit natural target cell retention as a result of theircontainment in high charge density chelates.

Evidence exists that streptavidin is resistant to metabolic degradation.Consequently, radionuclide bound directly or indirectly to streptavidin,rather than, for example, directly to the targeting moiety, are retainedat target cell sites for extended periods of time.Streptavidin-associated radionuclides can be administered inpretargeting protocols intravenously, intraarterially or the like orinjected directly into lesions.

U.S. Pat. No. 4,867,962 issued to Abrams describes an improved methodfor delivering active agent to target sites, which method employs activeagent-targeting moiety conjugates. Briefly, the Abrams methodcontemplates administration to a recipient of two or more activeagent-targeting moiety conjugates, wherein each conjugate includes atargeting moiety of a different antibody species. Each of the utilizedantibody species is reactive with a different target site epitope(associated with the same or a different target site antigen), and thepatterns of cross-reactivity for the antibody species arenon-overlapping. The active agent component of each administeredconjugate may be the same or different.

In this manner, the different antibodies (along with the agents attachedthereto) accumulate additively at the desired target site, while onlyone or fewer than the total administered antibody species accumulate oneach type of cross-reactive non-target tissue. A higher percentage ofthe administered agent therefore becomes localized in vivo at targetsites compared to non-target tissues. For diagnostic agents, thismethodology results in more clearly detected or imaged target sitesagainst a comparatively lower (i.e., more diffuse background ofnon-target tissue accumulation. Lower accumulation of therapeutic agentsat different non-target tissues permits larger doses of the associatedactive agents to be administered without incidence of undesirablenon-target toxicity.

The present invention provides two-step and three-step pretargetingmethods as set forth below.

The two-step approach involves:

administering to the recipient a first conjugate comprising a targetingmoiety of a first antibody species having a first pattern ofcross-reactivity and a member of a ligand-anti-ligand binding pair;

administering to the recipient one or more additional targetingconjugates, each such conjugate comprising a targeting moiety of adifferent antibody species from the species of the first conjugate andfrom each other and having a substantially non-overlapping pattern ofcross-reactivity from each other and from the first pattern ofcross-reactivity and the member of the ligand-anti-ligand pair bound tothe first conjugate;

optionally administering to the recipient a clearing agent capable ofdirecting the clearance of circulating conjugate from the recipient oroptionally treating the recipient with a clearing device or analternative clearing procedure to substantially remove circulatingconjugate from the recipient; and

administering to the recipient a second conjugate comprising an activeagent and a ligand/anti-ligand binding pair member, wherein the secondconjugate binding pair member is complementary to that of the firstconjugate.

One alternative to the optional clearance step set forth above is simplyto allow an amount of time to pass that is sufficient to permit therecipient's native clearance mechanisms to substantially removecirculating conjugate.

The three-step approach involves:

administering to the recipient a first conjugate comprising a targetingmoiety of a first antibody species and having a first pattern ofcross-reactivity and a ligand;

administering to the recipient one or more additional targetingconjugates, each such conjugate comprising a targeting moiety of adifferent antibody species from the species of the first conjugate andfrom each other and having a substantially non-overlapping pattern ofcross-reactivity from each other and from the first pattern ofcross-reactivity and the ligand bound to the first conjugate;

administering to the recipient an anti-ligand; and

administering to the recipient a second conjugate comprising the ligandand an active agent, wherein second conjugate localization at the targetsite is enhanced as a result of prior localization of the firstconjugate.

Alternatively, antibody-based or non-antibody-based targeting moietiesmay be employed to deliver a ligand or an anti-ligand to a target sitebearing an unregulated antigen. Preferably, a natural binding agent forsuch an unregulated antigen is used for this purpose. For example,diseases such as hepatoma or myeloma are generally characterized byunregulated IL-6 receptors for which IL-6 acts as an autocrine orparacrine moiety with respect to rapid proliferation of these targetcell types. For the treatment of such ailments, IL-6 may therefore beemployed as a targeting moiety in a pretargeting protocol of the presentinvention.

For example, IL-6 and streptavidin may be conjugated via chemical meansor be formed as a recombinant molecule. The IL-6-streptavidin conjugateis administered to a recipient, and the IL-6 component of the conjugatedirects the localization of the conjugate to IL-6 receptors. Thislocalization will occur preferentially to sites bearing unregulated IL-6receptors. After target site localization occurs, a clearing agent isoptionally administered to substantially clear the recipient'scirculation of IL-6-streptavidin conjugate. Suitable clearing agents forthis purpose are, for example, IL-6 receptor-HSA-galactose;anti-IL-6-antibody-HSA-galactose or the like. After a time sufficientfor substantial clearance of IL-6 from the recipient's circulation,active agent-biotin conjugate is administered and localizes to targetsites via the IL-6-streptavidin conjugate.

In the practice of these aspects of the present invention, the targetsite accretion of active agent conjugate receptor (i.e., the ligand oranti-ligand conjugated to the first antibody species and additionaltargeting antibody species) is improved, because each antibody speciesrecognizes a different epitope associated with the target site. Thisalternative epitope approach provides more potential target site bindingpoints for the active agent conjugate receptor. Consequently, actual oreffective target site saturation (via epitope saturation and/or sterichindrance) may be avoided; the target binding site barrier (binding ofsurface epitopes of the target site, which limits egress to internaltarget site epitopes) may be circumvented; and additive accumulation ofactive agent conjugate receptor may be accomplished. The practice ofthis aspect of the present invention provides a ligand- oranti-ligand-active agent conjugate with a target site that is denselypopulated with the anti-ligand or ligand antigen to which the activeagent-containing conjugate may bind. Up to a point dictated primarily bysteric factors, increased target site antigen density facilitatesincreased active agent accretion thereto.

The phrase "non-overlapping patterns of cross-reactivity" indicates thatthe non-target tissues bound by one antibody species differssubstantially from the non-target tissues bound by another antibodyspecies. The patterns of cross-reactivity must differ to the extentnecessary to proportionately reduce the background for diagnosticapplications and to reduce the toxicity to normal tissues fortherapeutic applications. The less overlap in cross-reactivity tonon-target tissues, the more useful an antibody pair (or larger set ofantibodies) in the practice of these aspects of the present invention.

The patterns of cross-reactivity for monoclonal antibodies directedagainst a particular target site are analyzed to identify a set of twoor more target-specific monoclonal antibodies with non-overlappingcross-reactivity for use in a diagnostic or therapeutic application.Antibodies may be screened by a variety of methods. The in vitroprocedure employed to determine reactivity with target tissue andcross-reactivity with non-target tissue is immunohistochemical analysis.Tissues to which the antibody species binds are identified by exposingthe tissue to the antibody; washing the tissue to remove any unboundantibody; and detecting the presence of bound antibody. Frozen tissuesections are preferred for use in these immunohistochemical methods,because tissue fixation may destroy epitopes and is associated withuncertainties in timing that may compromise antigen preservation. Forantigens that are known to be preserved following fixation, such atechnique may be effectively used. In vitro histochemical procedures areknown (e.g., Ceriani et al., Cancer Research, 47: 532-540, 1987 orExample I of U.S. Pat. No. 4,867,962).

In these multi-targeting moiety administering aspects of the presentinvention, the doses of each administered component will be determinedby the attending physician in accordance with his or her experience, theparticulars of the recipient's condition (the nature and location of thetarget site, including the antigens associated therewith, will impactantibody species selection and route of administration decisions) andthe combination of antibody species to be employed (antibody performancevaries with respect to antigen density and the affinity of the antibodyfor the antigen). It would be evident to one of ordinary skill in theart how to determine useful dosages of the components described above.

Another application of the present invention is in the area ofphotodynamic therapy. Photodynamic therapy itself is a two-stepprocedure as follows:

a photosensitizing agent that absorbs a certain wavelength of light istopically or systemically administered to the recipient and localizes tothe target site; and

target cells are illuminated with a light source of the appropriatewavelength. When the photosensitizing agent absorbs the light, ittransfers the absorbed energy to oxygen molecules dissolved in thetissue, thereby producing an active oxygen species which, in turn,destroys nearby biochemicals and, therefore, cells in the vicinity(primarily the target cells, provided that the photosensitizing agenthas selectively localized thereto).

The present invention provides pretargeting photodynamic therapyprotocols as set forth below.

The two-step approach involves:

administering to the recipient a first conjugate comprising a targetingmoiety and a member of a ligand-anti-ligand binding pair, wherein thefirst conjugate localizes to a target site;

optionally administering to the recipient a clearing agent capable ofdirecting the clearance of circulating conjugate from the recipient oroptionally treating the recipient with a clearing device or analternative clearing procedure to substantially remove circulatingconjugate from the recipient; and

administering to the recipient a second conjugate comprising aphotosensitizing agent and a ligand/anti-ligand binding pair member,wherein the second conjugate binding pair member is complementary tothat of the first conjugate and wherein the photosensitizing agent orthe conjugate as a whole is chemically modified to induce rapid and,preferably, renal clearance thereof from the recipient.

One alternative to the optional clearance step set forth above is simplyto allow an amount of time to pass that is sufficient to permit therecipient's native clearance mechanisms to substantially removecirculating conjugate.

The three-step approach involves:

administering to the recipient a first conjugate comprising a targetingmoiety and a ligand, wherein the targeting moiety-ligand conjugatelocalizes to a target site;

administering to the recipient an anti-ligand; and

administering to the recipient a second conjugate comprising the ligandand a photosensitive agent, wherein the photosensitizing agent or theconjugate as a whole is chemically modified to induce rapid and,preferably, renal clearance thereof from the recipient and whereinsecond conjugate localization at the target site is enhanced as a resultof prior localization of the first conjugate.

In both the two-step and the three-step approaches to photodynamictherapy set forth above, the final step is subjecting the recipient tolight of the appropriate wavelength. This step is preferably conductedbetween from about 2 hours to about 72 hours following administration ofthe photosensitizing agent-containing conjugate.

Optionally, an additional step of clearing the photosensitizingagent-containing conjugate may be employed. In this manner, rapidclearance of that agent may be facilitated. This clearance step may beachieved by administration of anti-ligand or galactosylated anti-ligand,for example, when photosensitizing agent-ligand conjugates are employedand by administration of ligand-HSA-galactose, for example, whenphotosensitizing agent-anti-ligand conjugates are employed.

To achieve target cell destruction, the photosensitizing agent is,preferably, selectively taken up by target cells. In the practice of thepresent invention, selective target site accretion is primarilyfacilitated by conjugating the photosensitizing agent to a ligand or ananti-ligand that binds a pretargeted anti-ligand or ligand with highaffinity. Route of administration can also impact photosensitizing agentaccretion, e.g., intraarterial administration for arterially accessibletarget sites.

Also, it is preferred that the photosensitizing agent is retained attarget cell sites for a period of time. Target site retention may beimparted by the targeting moiety through which the photosensitizingagent is associated with the target cell, if any, with release of thephotosensitizing agent from association with the targeting moiety overtime by the use of cleavable linkers or like methods; by target cellbiochemistry (i.e., photosensitizing agents soluble at low pH will beretained longer by target cells exhibiting low pH such as tumor cells);or by the hydrophobicity of the photosensitizing agent (greaterhydrophobicity enhances retention).

Preferred photosentizing agents for use in pretargeting protocols of thepresent invention directed at destroying target cells that are notadjacent or closely adjacent to the skin of the recipient also exhibitthe ability to absorb light of longer wavelengths. The longer thewavelength of light, the deeper that light can penetrate tissue.Consequently, photosensitizing agents that absorb longer wavelengths(e.g., between about 600 and about 800 nm) can act on target sitesembedded more deeply in tissue than photosensitizing agents absorbinglight of lower wavelengths. While initially only skin cancers weretreated with photodynamic therapy, the conventional procedure has nowbeen applied to early stage tumors in the head and neck, brain, lung,gastrointestinal and genitourinary tracts.

In addition, preferred photosensitizing agents for use in the practiceof the present invention are capable of efficiently producing highlyactive oxygen species. Generally, photosensitizing agents exhibitinggreater hydrophobicity are more efficient at producing highly activeoxygen species than such agents exhibiting lesser hydrophobicity. Thisgreater efficiency appears to be related to the greater ability ofhydrophobic moieties to penetrate cell membranes.

Common photosensitizing agents are porphyrin derivatives with a strongabsorption band between 600 and 700 nm (red light). Chemicalmodification of porphyrin compounds is undertaken to enhance performanceof those compounds in photodynamic therapy protocols. Phthalocyanines,synthetic porphyrins when chelated with aluminum or zinc (e.g.,chloroaluminum sulfonated phthalocyanines), are effective to destroytarget cells. Photofrin II, an ether/ester derivative of porphyrin, ispresently the most commonly employed photosensitizing agent inphotodynamic therapy. Other exemplary photosensitizing agents arechlorins (e.g., chlorin e6, tin chlorin e6, bacteriochlorin A,bacteriochlorophyllin a, mono- and di-L-aspartyl chlorin e6, and thelike); porphyrin diethers (e.g., di-isobutyl ethers and di-hexylethers); purpurins (e.g., NT2), benzoporphyrin derivatives (porphines,such as isomers of 5, 10, 15, 20-tetra(hydroxyphenyl)-porphyrin; andsulfonated derivatives of tetraphenylporphine, such as TPPS₂ (aderivative of TPPS₄ with two rather than four sulfonate groups) andTPPS₄ (5,10,15,20,-tetra(4-sulfonato-phenyl)-21H,23H,porphine)); and thelike. Generally, these exemplary photosensitizing agents havecarboxylate groups available for conjugation.

Any light source can be employed to activate the photosensitizingagents, provided it has the appropriate spectral characteristics.Various types of laser lights are being used for this purpose. A lasercan be coupled to a fiber-optic cable to deliver light precisely to therecipient without any energy loss. Other techniques, such aschemiluminescence can be used for local delivery of high intensitylight.

In addition, light sources encountered by recipients in their normalactivities (e.g., direct sunlight) also cause photosensitizing agents toproduce active oxygen molecules. This is one of the limitations ofconventional photodynamic therapy. Photosensitizing agents have longhalf-lives, generally up to about two months. A new benzoporphyrinderivative has exhibited a clearance time of about one week. In eithereven, recipients must avoid direct sunlight for the relevant time periodto avoid non-target tissue toxicity as the photosensitizing agent iscleared.

Photosensitizing agents of the present invention and methods of usingthem facilitate target cell-specific accretion of photosensitizing agentand obviate the necessity for a recipient avoiding direct sunlight. Whenthe agents and protocols of the present invention are employed, therecipient may even obtain a residual benefit from exposure to suchsunlight.

By decoupling the slow target site accretion of a targeting moleculefrom the kinetics of photosensitizing agent accretion and by utilizing ahigh affinity ligand-anti-ligand system to rapidly capturephotosensitizing agent conjugate, that active agent can quickly andspecifically accrete to the target site. The long circulating half-lifeof the photosensitizing agent renders this process somewhat inconvenientfor the recipient. Chemical modification of the photosensitizing agentto facilitate rapid excretion from the recipient would obviate thisdifficulty. Rapid and, preferably, renal excretion of thephotosensitizing agent-containing conjugate would permit the recipientto undertake normal activities within a few hours followingphotosensitizing agent administration. At that time, substantially allof the photosensitizing agent remaining in the recipient's system islocated at the target site. Consequently, exposure of the target to thedirected light source could be followed essentially immediately with theexposure of the recipient to an ambient light source of a wavelengthappropriate for producing activated oxygen with a substantially reducedrisk of non-target toxicity.

Photosensitizing agents may be coupled to ligands or anti-ligands inaccordance with known techniques. For example, porphyrin biotinylationmay be undertaken as set forth below. The carboxylic acid functionalityof porphyrin derivatives is activated by reaction withhydroxy-benztriazole. Biocytin or biocytin analogs are reacted withporphyrin benztriazole active ester in a biocytin:porphyrin molar ratioranging from about 2 to about 4. Porphyrins so derivatized contain 1 to3 biotins per porphyrin. Biotin conjugation is evaluated using the(μ-hydroxybenzene) benzoic acid (HABA) displacement assay employingpronase digested biotinylated porphyrin. Journal of BiologicalChemistry, 94:23C-24C, 1965.

Chemical modifications employed in the present invention are those thatfacilitate rapid excretion of photosensitizing agent-containingconjugates from the recipient. Preferably, such modifications alsodirect the photosensitizing agent-containing conjugate to renalexcretion. Appropriate chemical modifications may be made to thephotosensitizing agent or to the photosensitizing agent-containingconjugate. Preferably, the photosensitizing agent-conjugate may betreated with anion-forming agents as described below.

Useful anion-forming agents include compounds incorporating an anhydrideand/or at least one COOH group, such as succinic anhydride, other cyclicacid anhydrides, phthalic anhydride, maleic anhydride, N-ethyl maleimidesubstituted with carboxyl groups, aliphatic anhydrides (e.g., aceticanhydride), aromatic anhydrides, pH-reversible anhydrides (e.g.,citraconic anhydride and dimethyl maleic anhydride), alpha halo acidssuch as bromoacetate and iodoacetate, and diacids or triacidssubstituted with a functional group that reacts with a functional groupof a molecule to be charge-modified.

For example, succinic anhydride is dissolved in DMSO or another dryorganic solvent at a concentration of 40 mg per 200 microliters. Thissuccinic anhydride solution (or a dilution thereof up to 2.5 ml inanhydrous DMSO, 1.73×10⁻² M) is added, for example, to a protein (e.g.,antibody, antibody fragment, ligand, anti-ligand or conjugate containingone or more of these components) solution (e.g., 3-5 mg/ml incarbonate/bicarbonate buffer, pH 8.5-9.0) at molar ratios of succinicanhydride to protein of 1:5, 1:10 or 1:25. The reaction is carried outat room temperature for 15-30 minutes. After reaction completion,succinic acid is removed by ultrafiltration or by gel filtration. Thedegree of isoelectric shift is determined by isoelectric focusing.

The ability of charge-modified ligands and charge-modified anti-ligandsto bind to the complementary member of the ligand/anti-ligand pair istested in accordance with know procedures for testing ligand/anti-ligandbinding affinity.

For example, the photosensitizing agent-containing conjugates may bemodified by derivatization with a biodistribution directing moleculesuch as the following: hydrophilic polymers, such as 10 kD dextran,larger dextran molecules (having molecular weights ranging from about 20to about 70 kD), polyglutamates (having molecular weights ranging fromabout 5 to about 50 kD), succinylated polylysines (having molecularweights ranging from about 5 to about 50 kD) and definedoligosaccharides, i.e., oligosaccharides produced synthetically suchthat the structure is chemically defined and of sufficient size tosubstantially overcome liver and other organ uptake which facilitatesrenal excretion via glomular filtration. Polymers, such as thosedescribed above, are removed from recipients by renal excretion and,therefore, polymeric derivatization facilitates renal excretion ofpolymer-containing conjugate.

Biotin-polymer-porphyrin conjugates may be formed using commerciallyavailable biotinylated, lysine-derivatized dextran polymer (e.g.,biotin-dextran, lysine fixable available from Sigma Chemical Co., St.Louis, Mo.). The lysine derivatized-biotin is conjugated via thereaction of a lysine epsilon-amino group, for example, with thebenztriazole activated ester of the porphyrin described above. Otheractive esters as are employed in the art may also be used for thispurpose (e.g., N-hydroxysuccinimide; phenols substituted with strongelectron withdrawing groups such as nitro and fluoro groups; and thelike). Alternatively, biotin-polymer-porphyrin/chlorin conjugates, forexample, may be formed using commercially available biotinylated,lysine-derivatized dextran polymer that is activated by reaction of alysine residue thereof with the bifunctional reagent,succinimidyl-4-(N-maleimido-methyl)cyclohexane-1-carboxylate (SMCC),under conditions analogous to the porphyrin biotinylation set forthabove (e.g., pH, molar ratio and like conditions). The SMCC-derivatizeddextran polymer now contains the reactive maleimide functional groupavailable for conjugation with the thio analog of 5, 10, 15,20-tetra-(4-hydroxyphenyl)porphine, for example. This porphine isprepared from the corresponding p-hydroxyphenylporphine using syntheticprocedures that are known in the art.

Derivatization alternatives include (DTPA)_(N) where n ranges from about1 to about 2. DTPA, diethylene triamine penta-acetic acid, e.g., DTPAcyclic anhydride, may be linked by an amide bond via a nativecarboxylate group or a synthetically added carboxylate group of aphotosensitizer.

Another derivatization alternative is a hydrazine analog of5,10,15,20-tetra-(4-hydroxyphenyl)porphine which is prepared from thecorresponding p-hydroxyphenylporphine via the p-chlorophenylporphineintermediate using conventional synthetic procedures. Periodate-oxidizedbiotinylated dextran contains reactive aldehyde functional groups forconjugation with the hydrazine analog.

Sustained release dosage forms may also be employed in the process ofthe present invention to deliver photosensitizing agent to target cellsthrough the pretargeting approach. In this manner, the therapeuticeffect of the photosensitizing agent may be achieved over a period oftime. Ligand or anti-ligand derivatized liposomes may be employed forthis purpose. Hawrot et al., U.S. Pat. No. 4,948,590, for example,discuss streptavidinylated liposomes and the encapsulation of activeagents therein.

Alternatively, microparticulate or nanoparticulate polymeric bead dosageforms may be employed, such as those discussed in Example IX herein foruse as clearing agents. In this case, the active agent will beencapsulated in the particulate dosage forms which have a number ofligand or anti-ligand molecules bound thereon. In this manner, activeagent is delivered to a target site via ligand-anti-ligand binding andactive agent is release at that site over time to provide a sustainedtherapeutic benefit.

In general, the procedure for forming particulate dosage forms of thepresent invention involves dissolving the polymer in a halogenatedhydrocarbon solvent, dispersing an active agent solution (preferablyaqueous) therein, and adding an additional agent that acts as a solventfor the halogenated hydrocarbon solvent but not for the polymer. Thepolymer precipitates out from the polymer-halogenated hydrocarbonsolution onto droplets of the active agent containing solution andentraps the active agent. Preferably the active agent is substantiallyuniformly dispersed within the sustained release dosage form of thepresent invention. Following particulate formation, they are washed andhardened with an organic solvent. Water washing and aqueous non-ionicsurfactant washing steps follow, prior to drying at room temperatureunder vacuum.

For biocompatibility purposes, particulate dosage forms, characterizedby an active agent dispersed therein in matrix form, are sterilizedprior to packaging, storage or administration. Sterilization may beconducted in any convenient manner therefor. For example, theparticulates can be irradiated with gamma radiation, provided thatexposure to such radiation does not adversely impact the structure orfunction of the active agent dispersed in the therapeutic agent-polymermatrix or the ligand or anti-ligand attached thereto. If the activeagent, ligand or anti-ligand is so adversely impacted, the particulatedosage forms can be produced under sterile conditions.

Release of the active agent from the particulate dosage forms of thepresent invention can occur as a result of both diffusion andparticulate matrix erosion. Biodegradation rate directly impacts activeagent release kinetics. The biodegradation rate is regulable byalteration of the composition or structure of the sustained releasedosage form. For example, alteration of the lactide/glycolide ratio inpreferred dosage forms of the present invention can be conducted, asdescribed by Tice et al., "Biodegradable Controlled-Release ParenteralSystems," Pharmaceutical Technology, pp. 26-35, 1984; by inclusion ofpolymer hydrolysis modifying agents, such as citric acid and sodiumcarbonate, as described by Kent et al., "Microencapsulation of WaterSoluble Active Polypeptides," U.S. Pat. No. 4,675,189; by altering theloading of active agent in the lactide/glycolide polymer, thedegradation rate being inversely proportional to the amount of activeagent contained therein, and by judicious selection of an appropriateanalog of a common family of active agents that exhibit differentpotencies so as to alter said core loadings; and by variation ofparticulate size, as described by Beck et al.,"Poly(DL-Lactide-Co-Glycolide)/Norethisterone Microcapsules: AnInjectable Biodegradable Contraceptive," Biol. Reprod., 28:186-195,1983, or the like. All of the aforementioned methods of regulatingbiodegradation rate influence the intrinsic viscosity of the polymercontaining matrix, thereby altering the hydration rate thereof.

The preferred lactide/glycolide structure is biocompatible with themammalian physiological environment. Also, these preferred sustainedrelease dosage forms have the advantage that biodegradation thereofforms lactic acid and glycolic acid, both normal metabolic products ofmammals.

Functional groups required for ligand or anti-ligand bonding to theparticles, are optionally included in the particulate structure, alongwith the non-degradable or biodegradable polymeric units. Functionalgroups that are exploitable for this purpose include those that arereactive with ligands or anti-ligands such as carboxyl groups, aminegroups, sulfhydryl groups and the like. Preferred functional groupsinclude the terminal carboxyl groups of the preferred(lactide-glycolide) polymer containing matrix or the like.

These sustained release dosage forms are also useful with regard toother active agents useful in the practice of the present invention,such as toxins, chemotherapeutic agents, cytokines and the like.

Another strategy that may be employed to increase photosensitizerpotency and allow bystander cells to be treated involves the use ofcleavable linkers between the biotin or the biotin-biodistributiondirecting molecule and the photosensitizing agent. The advantage of thecontrolled instability offered by a cleavable linker is that morehydrophobic photosensitizers could be used. Photosensitizing agentsexhibiting enhanced hydrophobicity appear to have increased potency ofcell killing when exposed to light of the appropriate wavelength.Because of the rapid targeting and clearance of the photosensitizingagent-containing conjugate, cleavable linkers exhibiting a stabilityhalf-life under physiological conditions of several hours (e.g., betweenfrom about 3 to about 12 hours) and practical shelf life (e.g., betweenfrom about 3 months to about 2 years) may be employed. Exemplary of suchcleavable linkers are disulfide linkages, carboxylate ester linkagessuch as cis-aconitates, hydrazide linkages and the like. Herman et al.,Bioconjugate Chemistry, 4: 402-405, 1993, for example, describes thesynthesis dextran derivatives with thio-specific reactive groups. Suchdextran derivatives can be employed in forming conjugates bearing acleavable disulfide linkage. A hydrazide-linked conjugate may be formedin accordance with the procedure set forth in PNAS, 87: 4217-4221, 1990.

Monovalent antibody fragment-anti-ligand or -ligand conjugates may beemployed in pretargeting protocols of the present invention. Forexample, a monovalent antibody fragment-streptavidin conjugate may beused to pretarget streptavidin, preferably in additional embodiments ofthe two-step aspect of the present invention. Exemplary monovalentantibody fragments useful in these embodiments are Fv, Fab, Fab' and thelike. Monovalent antibody fragments, typically exhibiting a molecularweight ranging from about 25 kD (Fv) to about 50 kD (Fab, Fab'), aresmaller than whole antibody and, therefore, are generally capable ofgreater target site penetration. Moreover, monovalent binding can resultin less binding carrier restriction at the target surface (occurringduring use of bivalent antibodies, which bind strongly and adhere totarget cell sites thereby creating a barrier to further egress intosublayers of target tissue), thereby improving the homogeneity oftargeting.

In addition, smaller molecules are more rapidly cleared from arecipient, thereby decreasing the immunogenicity of the administeredsmall molecule conjugate. A lower percentage of the administered dose ofa monovalent fragment conjugate localizes to target in comparison to awhole antibody conjugate. The decreased immunogenicity may permit agreater initial dose of the monovalent fragment conjugate to beadministered, however.

A multivalent, with respect to ligand, moiety is preferably thenadministered. This moiety also has one or more radionuclide associatedtherewith. As a result, the multivalent moiety serves as both a clearingagent for circulating anti-ligand-containing conjugate (throughcross-linking or aggregation of conjugate) and as a therapeutic agentwhen associated with target bound conjugate. In contrast to theinternalization caused by cross-linking described above, cross-linkingat the tumor cell surface stabilizes the monovalent fragment-anti-ligandmolecule and, therefore, enhances target retention, under appropriateconditions of antigen density at the target cell. In addition,monovalent antibody fragments generally do not internalize as dobivalent or whole antibodies. The difficulty in internalizing monovalentantibodies permits cross-linking by a monovalent moiety serves tostabilize the bound monovalent antibody through multipoint binding. Thistwo-step protocol of the present invention has greater flexibility withrespect to dosing, because the decreased fragment immunogenicity allowsmore streptavidin-containing conjugate, for example, to be administered,and the simultaneous clearance and therapeutic delivery removes thenecessity of a separate controlled clearing step.

A potential difficulty in employing two-step and three-step pretargetingprotocols involving the biotin-avidin or the biotin-streptavidinligand-anti-ligand pair is the presence of endogenous biotin. Biotin isalso known as vitamin H and is present at an endogenous level inmammalian recipients. Mice, for example, have high levels of endogenousbiotin, ranging from about microgram to about nanogram concentrations(10⁻⁶ to 10⁻⁹ M). Larger mammals, such as rabbits and dogs, exhibitendogenous biotin at lower levels than mice, ranging from about lownanogram to about high nanogram concentrations (10⁻⁹ to 10⁻¹¹ M). Humansexhibit even lower endogenous biotin levels, ranging from about lowpicogram to about high picogram concentrations (10⁻¹² to 10⁻¹⁴ M).Because endogenous biotin level is impacted by factors such as diet andsuppression of gut wall bacteria by oral antibotics, variability inendogenous biotin level will be observed within each mammalian species.

While the two-step and three-step pretargeting methods of the presentinvention may be conducted despite endogenous biotin, methods ofdecreasing endogenous biotin or the impact thereof would be useful. Oneway to diminish the impact of endogenous biotin is to overwhelm theendogenous biotin with a high dose of targeting moiety-streptavidin or-avidin conjugate. More specifically, conjugate is administered in anamount sufficient to substantially bind both the endogenous biotin andsufficient target site epitopes to achieve a diagnostic or therapeuticbenefit for the recipient. In the identification of an appropriateconjugate dose, the endogenous biotin level for each individualrecipient may be determined, and the conjugate dose selectedaccordingly. Alternatively, an appropriate conjugate dose may be basedupon average endogenous biotin values for the recipient species.

Another method involves a pretreatment with an amount of avidinsufficient to bind up substantially all of a recipient's endogenousbiotin. In this method, avidin may be administered intravenously as abolus dose followed by slow infusion (e.g., avidin in saline or in PBS),preferably from about 5 minutes to about 30 minutes prior to targetingmoiety-streptavidin administration. Alternatively, avidin may beadministered orally (e.g., as raw egg whites), preferably earlier and athigher doses than an intravenous administration. Still anotheralternative is administering a high dose of avidin by enema (e.g.,avidin in saline), preferably earlier and at higher doses thanintravenous administration.

An intravenously administered agent becomes bioavailable faster than anagent administered via oral or enema routes, therefore generallyrendering intravenous administered agents more toxic than the agentsadministered by oral/enema routes. Also, absorption, distribution,kinetics and metabolism of oral/enema administrations are generallyslower than intravenous agent administrations, thereby generallyrequiring a higher dose for oral/enema administrations.

Alternatively, the recipient may be placed on a biotin-free diet priorto conducting a two-step or three-step pretargeting protocol. Forexample, a mouse recipient of a pretargeting protocol may be placed on abiotin-free diet from about 2 to about 3 days prior to the start of thepretargeting protocol. A larger volume mammal, such as a rabbit or adog, may be placed on a biotin-free diet from about 3 to about 7 daysbefore the first conjugate administration of a pretargeting protocol ofthe present invention. A human recipient may be placed on a biotin-freediet (e.g., avoiding dietary sources of biotin such as eggs, nuts,peanut butter, chocolate, candy, yeast, cereals, organ meats such askidney and liver, meat, meat products, mushrooms, bananas, grapefruit,watermelon, strawberries, beans and legumes) from about 1 to about 2weeks prior to commencement of the pretargeting protocol. In thismanner, the endogenous biotin level of the recipient may be reduced,thereby diminishing any adverse impacts of endogenous biotin on thepretargeting protocol.

An alternative method to address endogenous biotin is the use of oral,non-absorbable antibiotics. Most human endogenous biotin is produced bygut flora (e.g., bacteria, such as E. coli and the like). Potentantibiotics are known which destroy gut flora. Such antibiotics areorally administered and are not absorbed from the intestinal tract, sothat they are non-toxic to the recipient. Other functionalcharacteristics of suitable antibiotics are as follows: antagonism ofgrowth and/or survival of one or more species of microorganisms thatproduce biotin; effectiveness at low doses;and the like. Exemplary ofsuch antibiotics are ampicillin, chloramphenicol, erythromycin,oxacillin, nafcillin, oxytetracycline, penicillin-G, penicillin-V,tetracycline, kanamycin, lincomycin, griseofulvin, doxycycline,novobiocin, colistin, chlortetracycline, and the like. To temporarilylower the level of biotin produced by gut flora, oral, non-absorbableantibiotics, such as gentamicin, polymyxin-B, vancomycin and the like,may be administered from about 7 to about 10 days prior to thecommencement of the pretargeting protocol.

Combinations of the aforementioned methods may be employed in thepractice of the present invention. Of the above methods, all may beemployed in combination with two-step pretargeting.

Another embodiment of the pretargeting methodologies of the presentinvention involves the route of administration of the ligand- oranti-ligand-active agents. In these embodiments of the presentinvention, the active agent-ligand (e.g., radiolabeled biotin) or-anti-ligand is administered intraarterially using an artery supplyingtissue that contains the target. In the radiolabeled biotin example, thehigh extraction efficiency provided by avidin-biotin interactionfacilitates delivery of very high radioactivity levels to the targetcells, provided the radioactivity specific activity levels are high. Thelimit to the amount of radioactivity delivered therefore becomes thebiotin binding capacity at the target (i.e., the amount of antibody atthe target and the avidin equivalent attached thereto).

For these embodiments of the pretargeting methods of the presentinvention, particle emitting therapeutic radionuclide resulting fromtransmutation processes (without non-radioactive carrier forms present)are preferred. Exemplary radionuclides include Y-90, Re-188, At-211,Bi-212 and the like. Other reactor-produced radionuclides are useful inthe practice of these embodiments of the present invention, if they areable to bind in amounts delivering a therapeutically effective amount ofradiation to the target. A therapeutically effective amount of radiationranges from about 1500 to about 10,000 cGy depending upon severalfactors known to nuclear medicine practitioners.

Intraarterial administration pretargeting can be applied to targetspresent in organs or tissues for which supply arteries are accessible.Exemplary applications for intraarterial delivery aspects of thepretargeting methods of the present invention include treatment of livertumors through hepatic artery administration, brain primary tumors andmetastases through carotid artery administration, lung carcinomasthrough bronchial artery administration and kidney carcinomas throughrenal artery administration. Intraarterial administration pretargetingcan be conducted using chemotherapeutic drug, toxin and anti-tumoractive agents as discussed below. High potency drugs, lymphokines, suchas IL-2 and tumor necrosis factor, gamma-interferon,drug/lymphokine-carrier-biotin molecules, biotinylateddrugs/lymphokines, and drug/lymphokine/toxin-loaded, biotin-derivatizedliposomes are exemplary of active agents and/or dosage forms useful forthe delivery thereof in the practice of this embodiment of the presentinvention.

In embodiments of the present invention employing radionuclidetherapeutic agents, the rapid clearance of nontargeted therapeutic agentdecreases the exposure of non-target organs, such as bone marrow, to thetherapeutic agent. Consequently, higher doses of radiation can beadministered absent dose limiting bone marrow toxicity. In addition,pretargeting methods of the present invention optionally includeadministration of short duration bone marrow protecting agents, such asWR 2721, as well as other protective agents such as IL-3, GM-CSF, G-CSFor a combination of IL-3 and GM-CSF. As a result, even higher doses ofradiation can be given, absent dose limiting bone marrow toxicity.

Also, pretargeting techniques are generally characterized by relativelyrapid target site accretion of active agent, because of the highaffinity binding between the members of a ligand-anti-ligand pair. Forexample, active agent accretion has reached a therapeutically ordiagnostically significant level within from about 1 to about 8 hoursfollowing administration of active agent-containing conjugate.Consequently, radionuclides having appropriately short half-lives may beemployed in pretargeting protocols of the present invention. In thismanner, non-target toxicity is further reduced. Exemplary shorthalf-life radionuclides include Cu-64, Cu-67, Lu-177, Rh-105, I-123,I-131, Sm-153, Re-186, Re-188, Bi-212, Pb-212, At-211, Y-90, In-111,Tc-99m and the like.

While the pretargeting protocols set forth above have been describedprimarily in combination with delivery of a radionuclide diagnostic ortherapeutic moiety, the protocols are amenable to use for delivery ofother moieties, including anti-tumor agents, chemotherapeutic drugs andthe like. For example, most naturally occurring and recombinantcytokines have short in vivo half lives. This characteristic limits theclinical effectiveness of these molecules, because near toxic doses areoften required. Dose-limiting toxicities in humans have been observedupon high dose IL-2 or tumor necrosis factor, gamma-interferon orlymphotoxin administrations, for example.

Anti-tumor agents, such as IL-2 and TNF, may be employed as activeagents in the practice of two-step or three-step pretargeting protocolsof the present invention. Some anti-tumor agents exhibit shortcirculation half-lives (less than about 1 hour post-administration),such as IL-2 (half-life of about 10 minutes), other interleukins, TNF,interferons and the like. Such short half-life active agents areamenable to use in pretargeting protocols of the present invention.

Anti-tumor agents having longer half-lives (ranging from about 2 hoursto about 12 hours post-administration) are preferably employed at lowdoses, while conjugates incorporating such moieties at high dosespreferably also incorporate a biodistribution directing moiety, such asa polymer, to direct the biodistribution of the conjugate and activeagent-containing metabolites thereof to renal excretion. Ligand oranti-ligand derivatization of a long half-life anti-tumor agent maydecrease the serum half-life sufficiently to permit higher doses ofconjugate to be administered, however.

IL-2 has a molecular weight of 15,500 daltons and is formed of 133 aminoacids (42% nonpolar and 58% polar). IL-2 is characterized by 12 freeprimary amines for reaction with a ligand or an anti-ligand (e.g.,biotin or streptavidin) or with functional groups of a biodistributiondirecting molecule (e.g., polymer). A single disulfide Cys58-Cys105appears essential for activity. The Cys125 residue appears not to berequired for some uses and provides an additional functional group forderivatization. The three dimensional crystal structure of IL-2 has beensolved to 3 angstrom resolution. Most of the secondary structure isalpha helical in nature. Three of the alpha helices (residues 11-19;residues 33-56; and residues 107-113) appear to be important for IL-2binding and activity. Five of the twelve free amines are located inthese helices, and, preferably, derivatization of IL-2 is not conductedvia these amines. IL-2 is normally purified by mono-S and reverse phasehigh pressure liquid chromatography (RP). Elution conditions for RP (60%acetonitrile, pH 2-3) suggest a very stable or, at least, an elasticmolecule. Consequently, IL-2 appears to be amenable to a number ofconjugation techniques.

IL-2-biotin conjugates may be formed using biotinamidocaproateN-hydroxysuccinimide ester (commercially available from Sigma ChemicalCo.). The reaction of IL-2 and biotinamidocaproate N-hydroxysuccinimdeester is conducted at room temperature for 0.5 hours in a 0.1M sodiumborate buffer, pH 8.0-8.5, containing 0.1% sodium dodecyl sulfate tokeep IL-2 in solution. When the IL-2 concentration is 5-10 mg/ml ofreaction buffer, biotin is incorporated at 75-80% when imidate ester,dissolved in DMSO in a volume no greater than 5-10% of the totalreaction volume, is added at a 2-4 biotin:IL-2 molar ratio. The productconjugate contains 1.5-3 biotins per IL-2 molecule. Biotin incorporationis assessed using the 2-(4-hydroxyazobenzene)benzoic acid (HABAdisplacement assay using pronase digested biotinylated IL-2.

Polymer-IL-2-biotin conjugates may be formed using commerciallyavailable biotinylated, lysine-derivatized dextran polymer (SigmaChemical Co.) that is activated by reacting a lysine residue thereofwith the bifunctional reagent SMCC, under conditions analogous to thebiotinylation of IL-2 set forth above (e.g., molar ratio and the like).The SMCC-derivatized dextran polymer now contains the reactive maleimidefunctional group available for conjugation with the cysteine sulfhydrylmoiety of IL-2 at pH 5-7 in acetate/phosphate buffer.

IL-2 receptor-bearing cells include activated T-cells, normal T-cells,activated B-cells, NK cells, LAK cells and thymocytes. High,intermediate and low affinity receptors for IL-2 exist. However, onlyabout 10% of the receptors for IL-2 appear to be high affinity receptors(K_(d) approximately 10⁻¹¹ M).

In the practice of a two-step pretargeting aspect of the presentinvention, IL-2 may be delivered to target cells as follows:

administering to the recipient a first conjugate comprising a targetingmoiety and a member of a ligand-anti-ligand binding pair, wherein thefirst conjugate localizes to a target site;

optionally administering to the recipient a clearing agent capable ofdirecting the clearance of circulating conjugate from the recipient oroptionally treating the recipient with a clearing device or analternative clearing procedure to substantially remove circulatingconjugate from the recipient; and

administering to the recipient a second conjugate comprising ananti-tumor agent, such as IL-2, and a ligand/anti-ligand binding pairmember, wherein the second conjugate binding pair member iscomplementary to that of the first conjugate.

One alternative to the optional clearance step set forth above is simplyto allow an amount of time to pass that is sufficient to permit therecipient's native clearance mechanisms to substantially removecirculating conjugate.

Concentrations of IL-2 effective in maintaining activated tumorinfiltrating lymphocytes (TIL) and for other forms of anti-tumor therapyhave also been found to be toxic to the recipient. Localization of IL-2to the tumor microenvironment allows localized activation of effectorcells to poorly immunogenic tumor antigens. Effector cells of thecytotoxic T-lymphocyte lineage and T-helper lineage are induced torecognize tumor antigens and clonally expand to seek out tumormetastases at other locations.

Furthermore, B cells may be induced to secrete antibody specificallyrecognizing tumor antigens. These B cells mature into IgG secretorycells and memory cells and continue to expand when tumor antigens aredetected at other sites. In addition, natural killer cells arecandidates for anti-tumor immunity, because such cells are alsoactivated by IL-2. Studies in SCID-beige mice deficient in T and B cellsbut NK competent showed that such mice were effective hosts in rejectingan IL-2 transfected tumor cell line. See Alosco et al., Cancer Immunol.Immunother., 36: 364-372, 1993.

Tumor necrosis factors (TNFs) have been isolated from a variety ofmammalian species. For example, human, murine, rabbit and guinea pigexhibit at least alpha and beta forms of TNF. In humans, TNF-alphaexhibits a molecular weight of 45 (gel filtration) and 17 (SDS-PAGE), anisoelectric point of 5.6, no glycosylation, protease sensitivity, 2cysteine residues, 157 amino acids and a terminal valine residue.TNF-beta exhibits a molecular weight of 65 (gel filtration) and 25(SDS-PAGE), an isoelectric point of 5.8, glycosylation, proteaseresistance, no cysteine residues, 171 amino acids, and a terminalleucine residue. The serum half-lives of TNF-alpha and TNF-beta areapproximately 10 to 15 minutes.

TNF-alpha and TNF-beta exhibit free primary amines for conjugation tothe ligand biotin, for example, or to a functional group of abiodistribution directing molecule. TNF-biotin conjugates may be formedas follows: Biotinamidocaproate N-hydroxysuccinimidate (dissolved in asmall volume of DMSO) is offered at a 4-8 biotin:TNF molar ratio withrespect to TNF (dissolved in 0.1M borate buffer, pH 8.0-8.5, at aprotein concentration of 0.5 mg/ml). After 2 hours at room temperatureTNF has incorporated 1 biotin per TNF molecule as assessed by the HABAassay.

Polymer-TNF-biotin conjugates may be formed using commercially availablebiotinylated, lysine-derivatized dextran polymer (10 kD-70 kD glucanpolymer supplied by Sigma Chemical Co.) that is reacted withN-succinimidyl-3-(2-pyridyl-dithio)propionate (SPDP), under conditionsanalogous to the biotinylation of TNF as described above to give rise toa pyridyl dithio-derivatized polymer. TNF is derivatized by a lysineresidue thereof with the bifunctional reagent SMCC, under conditionsanalogous to the biotinylation of TNF set forth above to give rise tomaleimidyl-derivatized TNF. To the pyridyl dithio polymer,dithiothreitol (DTT) is added in an oxygen free environment at a 2:1DTT:polymer molar ratio to generate a free sulfhydryl moiety on thepolymer. After purification of the derivatized polymer in the oxygenfree environment, the maleimidyl-derivatized TNF is added, wherein themaleimidyl group reacts with the sulfhydryl moiety of the derivatizedpolymer to form the product conjugate.

TNF receptor-bearing cells include adipocytes, myotubes, cervicalcarcinoma, fetal lung, bladder carcinoma, histocytic leukemia,erthroleukemia, promyelocytic leukemia, epidermoid carcinoma, cervicalcarcinoma, T lymphoma, human lymphocytes, lymphoblastic leukemia (tworeceptors), monocytic leukemia, foreskin fibroblast, connective tissue(two receptors), murine macrophage, and bovine endothelium. High,intermediate and low affinity receptors for TNF exist.

TNF-alpha and TNF-beta have been shown to exhibit different biologicaleffects on certain target cells, including endothelial cells (productionof IL-1), myeloid cells (clonogenic survival), fibroblasts andmacrophages (production of macrophage colony-stimulating factor),neutrophils (activation of neutrophils), osteoblasts (proliferation,release of Ca⁺² collagen degradation), vascular smooth muscle cells(interferon-gamma-dependent expression of human lymphocyte antigen-DR),T-cell hybridoma (MHC-I cell expression), and B lymphocytes (growthfactor).

In the practice of a two-step pretargeting aspect of the presentinvention, TNF may be delivered to target cells essentially in themanner described above for IL-2.

TNF itself is cytotoxic to a narrow spectrum of tumor cells; however,this cytokine exhibits a broad range of immunologic modulatingactivities. One such activity is activation of tumor infiltratingmacrophages or monocytes, thereby rendering the macrophages tumoricidal.One theory regarding the mechanism of TNF in this regard suggests thatTNF stimulates monocytes to progress to macrophages which are, in turn,stimulated by TNF to release cytotoxic factors (e.g., oxidative burst orprotease secretion or cytokine release). TNF release by activatedmacrophages can maintain or induce tumoricidal activity through anautocrine mechanism. Additional activation of monocytes or macrophagesby other cytokines (e.g., gamma-interferon) may be employed to enhancethe cytotoxic effect.

For example, membrane preparations from gamma-interferon-activatedmonocytes are cytotoxic to K562 (erythroleukemia cell line) or WEHI164(murine fibrosarcoma cell line) target cells. Pretreatment of monocyteswith recombinant TNF-alpha for 1 hour followed by treatment of mediumalone or gamma-interferon led to increased killing of the aforementionedtumor cell types by the TNF-alpha/gamma-interferon-treated membranepreparations of monocytes. See, for example, Peck et al., CellularImmunol., 132: 308, 1991).

Burrows et al., Proc. Natl. Acad. Sci. USA, 90: 8996-9000, 1993, discussthe concept of vascular targeting. Tumor target vascular endothelialcells are accessible to circulating agents. Burrows et al. transfected aneuroblastoma cell line with the murine INF-gamma gene. Thesetransfected cells as well as their non-transfected counterparts weregrown subcutaneously in BALB/c nu/nu mice. The transfected cellssecreted INF-gamma, which induced expression of class II antigens of themajor histocompatibility complex by capillary and venular endothelialcells within the tumor mass. An immunotoxin targeted to such MHC classII antigens was then administered and rapid accretion to tumor and tumorregressions were observed. Relapses were observed 7-10 days aftertreatment and were attributed to surviving tumor cells that derivednutrition from the extratumoral blood supply.

The pretargeting approach may be employed in conducting vasculartargeting diagnostic or therapeutic protocols. For example, a suitabletwo-step pretargeting method useful in practicing vascular targetingincludes the following:

administering to the recipient a first conjugate comprising a targetingmoiety specific for tumor endothelial cells and INF-gamma, wherein thefirst conjugate localizes to a target site and the INF-gamma inducesexpression of MHC class II antigens by tumor endothelial cells;

optionally administering to the recipient a clearing agent capable ofdirecting the clearance of circulating conjugate from the recipient oroptionally treating the recipient with a clearing device or analternative clearing procedure to substantially remove circulatingconjugate from the recipient; and

administering to the recipient a second conjugate comprising ananti-tumor agent, such as a toxin, a radionuclide, an anti-tumor agentor the like, and a targeting agent specific for MHC class II antigens.

An alternative two-step pretargeting method useful in vascular targetingis as follows:

administering to the recipient a first conjugate comprising a targetingmoiety specific for tumor endothelial cells and a member of aligand-anti-ligand binding pair, wherein the first conjugate localizesto a target site;

optionally administering to the recipient a clearing agent capable ofdirecting the clearance of circulating conjugate from the recipient oroptionally treating the recipient with a clearing device or analternative clearing procedure to substantially remove circulatingconjugate from the recipient; and

administering to the recipient a second conjugate comprising ananti-tumor agent, such as a toxin, a radionuclide or an anti-tumoragent, and a ligand/anti-ligand binding pair member, wherein the secondconjugate binding pair member is complementary to that of the firstconjugate. This latter protocol offers the advantages of not relying ontumor endothelial cells to express the antigen recognized by the activeagent-bearing conjugate and of not exposing the recipient tosystemically administered INF-gamma.

One alternative to the optional clearance step set forth above is simplyto allow an amount of time to pass that is sufficient to permit therecipient's native clearance mechanisms to substantially removecirculating conjugate.

Also, an optional additional step is the administration of a conjugateincorporating a targeting moiety specific for tumor cells and acytotoxic active agent. Alternatively, administration of a conjugatecomprising a targeting moiety specific for tumor cells and the member ofthe ligand-anti-ligand binding pair incorporated in the first conjugate,wherein this conjugate localizes to a target site. In this manner, tumorcells that receive nutrition from the extratumoral vasculature can beaddressed.

A protocol, such as administration of streptavidin-targeting moietyconjugate followed by administration of biotinylated cytokine, is alsocontemplated by the present invention. Such pretargeting of anti-ligandserves to improve the performance of cytokine therapeutics by increasingthe amount of cytokine localized to target cells.

Streptavidin-antibody conjugates generally exhibit pharmacokineticssimilar to the native antibody and localize well to target cells,depending upon their construction. Biotinylated cytokines retain a shortin vivo half-life; however, cytokine may be localized to the target as aresult of the affinity of biotin for avidin. In addition, biotin-avidinexperience a pH-dependent dissociation which occurs at a slow rate,thereby permitting a relatively constant, sustained release of cytokineat the target site over time. Also, cytokines complexed to target cellsthrough biotin-avidin association are available for extraction andinternalization by cells involved in cellular-mediated cytotoxicity.

A pre-formed antibody-streptavidin-biotin-cytokine preparation may alsobe employed in the practice of these methods of the present invention.In addition, a three-step protocol of the present invention may also beemployed to deliver a cytokine, such as IL-2, to a target site.

Other anti-tumor agents that may be delivered in accordance with thepretargeting techniques of the present invention are selecting,including L-selectin, P-selectin and E-selectin. The presence ofcytokines stimulates cells, such as endothelial cells, to expressselectins on the surfaces thereof. Selectins bind to white blood cellsand aid in delivering white blood cells where they are needed.Consequently, a protocol, such as administration of streptavidin- oravidin-targeting moiety conjugate followed by administration ofbiotinylated selecting, is also contemplated by the present invention.Such pretargeting of anti-ligand serves to improve the performance ofselectin therapeutics by increasing the amount of selectin localized totarget cells. In this manner, the necessity of cytokine induction ofselectin expression is obviated by the localization and retention ofselectin at a target cell population.

Chemotherapeutic drugs also generally exhibit short in vivo half-livesat a therapeutically effective dose. Consequently, another example of aprotocol of the present invention includes administration ofavidin-targeting moiety conjugate followed by administration of abiotin-chemotherapeutic drug conjugate or complex, such as adrug-carrier-biotin complex. A three-step protocol of the presentinvention may also be employed to deliver a chemotherapeutic drug, suchas methotrexate, adriamycin, high potency adriamycin analogs,trichothecenes, potent enediynes, such as esperamycins andcalicheamycins, cytoxan, vinca alkaloids, actinamycin D, taxol, taxotereor the like to a target site.

While the majority of the exemplary pretargeting protocols set forthabove have been directed to the treatment of cancer, pretargetingprotocols may be employed in the diagnosis or treatment of otherconditions. Exemplary additional conditions are discussed below.

For example, active agents that are cytotoxic to activated cytotoxicT-cells, such as trichothecenes (e.g., Roridin A, Verrucarin A,anguidine, scirpenetriol and the like) as well as agents that decreasehelper T-cell activity or helper T-cell number, such as TGF-beta, orincrease Ts activity, and the like will facilitate the treatment ofautoimmune diseases and transplantation rejection prevention. Tissuelesions in autoimmune disease are caused by the direct action ofactivated cytotoxic T-cells. Tissue transplantation is also, in part,mediated by the action of activated T-cells that bind to the cells ofthe new tissue and lyse them. Active agents that selectively de-activateor selectively kill activated cytotoxic T-cells can be employed asactive agents in pretargeting protocols of the present inventiondirected at the treatment of autoimmune disease or the prevention oftissue rejection. Such active agents may also be administered incombination with a second active agent that disrupts antibody (producedby B cells with the assistance of helper T-cells and IL-2)-complementmediated cell lysis associated with graft rejection.

Targeting to the target tissues may be accomplished via antibodiesdirected to the IL-2 receptor such as anti-TAC, anti-IL-2 receptorantibodies (e.g., anti-P-55 receptor and anti-P-75 receptor) or by IL-2itself or the like. IL-2 is rapidly internalized upon binding to itsreceptor and is otherwise primarily excreted via the renal pathway.Consequently, IL-2 would be a less desirable targeting moiety forpretargeting, as it may not remain on the surface of target cells longenough to allow completion of the protocol.

For example, delivery of active agents, such as interferon-gamma, tumornecrosis factor-alpha or a combination thereof, to monocytes ormacrophages or tumor cells via pretargeting protocols will facilitatethe treatment of cancer through the mechanism of activating themonocytes to cytotoxic macrophages. Activated macrophages excrete toxicmoieties such as enzymes, lysozyme cathepsins and hydrolases. Tumornecrosis factor-alpha acts via activation of cytotoxic macrophages,direct tumor cell killing or rendering the tumor cells more susceptibleto effector cell-mediated cytotoxicity. A combination of the two activeagents serves to optimally prime macrophage tumoricidal activity.Targeting to the target tissues may be accomplished via antibodiesdirected to tumor specific antigens, such that the active agents arepresented to the infiltrating monocyte population in a localizedenvironment.

For example, delivery of active agents, such as interleukin-2, tocytotoxic T-cells via pretargeting protocols will facilitate thetreatment of cancer through the mechanism of activating tumorinfiltrating lymphocytes (TIL). Killing by cytotoxic T-cells is believedto be mediated by release of pore-forming moieties that insert into themembrane of the target cell and facilitate target cell lysis. Targetingto the target tissues may be accomplished via antibodies directed totumor specific antigens.

For example, delivery of active agents, such as granulocyte monocytecolony stimulating factor (GM-CSF), to macrophages and polymorphonuclearneutrophils via pretargeting protocols will facilitate the treatment ofcancer through the mechanism of activating infiltrating monocytes andpolymorphonuclear leukocytes (PMNs). Killing by monocytes andpolymorphonuclear leukocytes is the result of cytotoxic enzymes releasedby activated forms of these cells. Targeting to the target tissues maybe accomplished via antibodies directed to tumor specific antigens.

For example, delivery of active agents, such as transforming growthfactor-beta (TGF-beta), to pancreatic tissue via pretargeting protocolswill facilitate the treatment of insulin-dependent diabetes mellitusthrough the mechanism of inhibition of cytotoxic T-cell maturation andinhibition of T-cell proliferation. Delivery to pancreatic tissue may beaccomplished, for example, an antibody or other targeting moiety whichrecognizes an antigen present on islet cells. Killing by T-cells is theresult of the release of pore-forming moieties by activated forms ofthese cells. Targeting to the target tissues may be accomplished viaantibodies directed to the pancreas.

TGF-beta may also be employed in the treatment of inflammatory diseaseusing pretargeting protocols of the present invention. Exemplary chronicinflammatory diseases which may be so treated are rheumatoid arthritis,thyroid disease in Hashimoto's thyroiditis, tuberculoid granuloma andthe like. TGF-beta is delivered to target by an antibody or othertargeting moiety which recognizes fibroblast activation protein orantigen 19 on stimulated stromal fibroblasts. See, for example, Rettiget al., Cancer. Res., 53: 3327, 1993. In this manner, TGF-beta may beused to inhibit chronic inflammation by targeting stromal fibroblastsactivated by peptide mediators and proteolytic enzymes.

For example, delivery of active agents, such as roridin A, verrucarin A,anguidine and like trichothecenes to RNA via pretargeting protocols willfacilitate the treatment of insulin-dependent diabetes mellitus orcancer through the mechanism of protein synthesis inhibition. Targetingto the target tissues may be accomplished via antibodies directed totumor specific antigens or to the pancreas.

An additional aspect of the present invention is directed to the use oftargeting moieties that are monoclonal antibodies or fragments thereofthat localize to an antigen that is recognized by the antibody NR-LU-10.Such monoclonal antibodies or fragments may be murine or of othernon-human mammalian origin, chimeric, humanized or human.

NR-LU-10 is a 150 kilodalton molecular weight IgG2b monoclonal antibodythat recognizes an approximately 40 kilodalton glycoprotein antigenexpressed on most carcinomas. In vivo studies in mice using an antibodyspecific for the NR-LU-10 antigen revealed that such antibody was notrapidly internalized, which would have prevented localization of thesubsequently administered active-agent-containing conjugate to thetarget site.

NR-LU-10 is a well characterized pancarcinoma antibody that has beensafely administered to over 565 patients in human clinical trials. Thehybridoma secreting NR-LU-10 was developed by fusing mouse splenocytesimmunized with intact cells of a human small cell lung carcinoma withP3×63/Ag8UI murine myeloma cells. After establishing a seed lot, thehybridoma was grown via in vitro cell culture methods, purified andverified for purity and sterility.

Radioimmunoassays, immunoprecipitation and Fluorescence-Activated CellSorter (FACS) analysis were used to obtain reactivity profiles ofNR-LU-10. The NR-LU-10 target antigen was present on either fixedcultured cells or in detergent extracts of various types of cancercells. For example, the NR-LU-10 antigen is found in small cell lung,non-small cell lung, colon, breast, renal, ovarian, pancreatic, andother carcinoma tissues. Tumor reactivity of the NR-LU-10 antibody isset forth in Table A, while NR-LU-10 reactivity with normal tissues isset forth in Table B. The values in Table B are obtained as describedbelow. Positive NR-LU-10 tissue reactivity indicates NR-LU-10 antigenexpression by such tissues. The NR-LU-10 antigen has been furtherdescribed by Varki et al., "Antigens Associated with a Human LungAdenocarcinoma Defined by Monoclonal Antibodies," Cancer Research, 44:681-687, 1984, and Okabe et al., "Monoclonal Antibodies to SurfaceAntigens of Small Cell Carcinoma of the Lung," Cancer Research, 44:5273-5278, 1984.

The tissue specimens were scored in accordance with three reactivityparameters: (1) the intensity of the reaction; (2) the uniformity of thereaction within the cell type; and (3) the percentage of cells reactivewith the antibody. These three values are combined into a singleweighted comparative value between 0 and 500, with 500 being the mostintense reactivity. This comparative value facilitates comparison ofdifferent tissues. Table B includes a summary reactivity value, thenumber of tissue samples examined and the number of samples that reactedpositively with NR-LU-10.

Methods for preparing antibodies that bind to epitopes of the NR-LU-10antigen are described in U.S. Pat. No. 5,084,396. Briefly, suchantibodies may be prepared by the following procedure:

absorbing a first monoclonal antibody directed against a first epitopeof a polyvalent antigen onto an inert, insoluble matrix capable ofbinding immunoglobulin, thereby forming an immunosorbent;

combining the immunosorbent with an extract containing polyvalentNR-LU-10 antigen, forming an insolubilized immune complex wherein thefirst epitope is masked by the first monoclonal antibody;

immunizing an animal with the insolubilized immune complex;

fusing spleen cells from the immunized animal to myeloma cells to form ahybridoma capable of producing a second monoclonal antibody directedagainst a second epitope of the polyvalent antigen;

culturing the hybridoma to produce the second monoclonal antibody; and

collecting the second monoclonal antibody as a product of the hybridoma.

Consequently, monoclonal antibodies NR-LU-01, NR-LU-02 and NR-LU-03,prepared in accordance with the procedures described in theaforementioned patent, are exemplary targeting moieties useful in thisaspect of the present invention.

Additional antibodies reactive with the NR-LU-10 antigen may also beprepared by standard hybridoma production and screening techniques. Anyhybridoma clones so produced and identified may be further screened asdescribed above to verify antigen and tissue reactivity.

                                      TABLE A                                     __________________________________________________________________________    TUMOR REACTIVITY OF ANTIBODY NR-LU-10                                         Organ/Cell Type                                                                            # Pos/                                                                            Intensity.sup.a                                                                      Percent.sup.b                                                                        Uniformity.sup.c                               Tumor        Exam                                                                              Avg.                                                                             Range                                                                             Avg.                                                                             Range                                                                             Avg.                                                                             Range                                       __________________________________________________________________________    Pancreas Carcinoma                                                                         6/6 3  3   100                                                                              100 2.3                                                                              2-3                                           Prostate Carcinoma 9/9 2.8 2-3 95 80-100 2 1-3                                Lung Adenocarcinoma 8/8 3 3 100 100 2.2 1-3                                   Lung Small Cell Carcinoma 2/2 3 3 100 100 2 2                                 Lung 8/8 2.3 2-3 73 5-100 1.8 1-3                                             Squamous Cell Carcinoma                                                       Renal Carcinoma 8/9 2.2 2-3 83 75-100 1 1                                     Breast Adenocarcinoma 23/23 2.9 2-3 97 75-100 2.8 1-3                         Colon Carcinoma 12/12 2.9 2-3 98 95-100 2.9 2-3                               Malignant Melanoma Ocular 0/2 0 0 0 0 0 0                                     Malignant Melanoma  0/11 0 0 0 0 0 0                                          Ovarian Carcinoma 35/35 2.9 2-3 200 100 2.2 1-3                               Undifferentiated 1/1 2 2 90 90 2 2                                            Carcinoma                                                                     Osteosarcoma 1/1 2 2 20 20 1 1                                                Synovial Sarcoma 0/1 0 0 0 0 0 0                                              Lymphoma 0/2 0 0 0 0 0 0                                                      Liposarcoma 0/2 0 0 0 0 0 0                                                   Uterine Leiomyosarcoma 0/1 0 0 0 0 0 0                                      __________________________________________________________________________     .sup.a Rated from 0-3, with 3 representing highest intensity                  .sup.b Percentage of cells stained within the examined tissue section.        .sup.c Rates from 0-3, with 3 representing highest uniformity.           

                  TABLE B                                                         ______________________________________                                        Organ/Cell Type   # Pos/Exam                                                                              Summary Reactivity                                ______________________________________                                        Adenoid                                                                         Epithelium          3/3       433                                             Lymphoid Follicle-Central 0/3 0                                               Lymphoid Follicle-Peripheral 0/3 0                                            Mucus Gland 2/2 400                                                         Adipose Tissue                                                                  Fat Cells           0/3       0                                             Adrenal                                                                         Zona Fasciculata Cortex                                                                           0/3       0                                               Zona Glomerulosa Cortex 0/3 0                                                 Zona Reticularis Cortex 0/3 0                                                 Medulla 0/3 0                                                               Aorta                                                                           Endothelium         0/3       0                                               Elastic interna 0/2 0                                                         Tunica Adventitia 0/3 0                                                       Tunica Media 0/3 0                                                          Brain-Cerebellum                                                                Axons, Myelinated   0/3       0                                               Microglia 0/3 0                                                               Neurons 0/3 0                                                                 Purkenje's Cells 0/3 0                                                      Brain-Cerebrum                                                                  Axons, Myelinated   0/3       0                                               Microglia 0/3 0                                                               Neurons 0/3 0                                                               Brain-Midbrain                                                                  Axons, Myelinated   0/3       0                                               Microglia 0/3 0                                                               Neurons 0/3 0                                                               Colon                                                                           Mucosal Epithelium  33        500                                             Muscularis Externa 0/3 0                                                      Muscularis Mucosa 0/3 0                                                       Nerve Ganglia 0/3 0                                                           Serosa 0/1 0                                                                Duodenum                                                                        Mucosal Epithelium  3/3       500                                             Muscularis Mucosa 0/3 0                                                     Epididymis                                                                      Epithelium          3/3       419                                             Smooth Muscle 0/3 0                                                           Spermatozoa 0/1 0                                                           Esophagus                                                                       Epithelium          3/3       86                                              Mucosal Gland 2/2 450                                                         Smooth Muscle 0/3 0                                                         Gall Bladder                                                                    Muscoal Epithelium  0/3       467                                             Smooth Muscle 0/3 0                                                         Heart                                                                           Myocardium          0/3       0                                               Serosa 0/1 0                                                                Ileum                                                                           Lymph Node          0/2       0                                               Mucosal Epithelium 0/2 0                                                      Muscularis Externa 0/1 0                                                      Muscularis Mucosa 0/2 0                                                       Nerve Ganglia 0/1 0                                                           Serosa 0/1 0                                                                Jejunum                                                                         Lymph Node          0/1       0                                               Mucosal Epithelium 2/2 400                                                    Muscularis Externa 0/2 0                                                      Muscularis Mucosa 0/2 0                                                       Nerve Ganglia 0/2 0                                                           Serosa 0/1 0                                                                Kidney                                                                          Collecting Tubules  2/3       160                                             Distal Convoluted Tubules 3/3 500                                             Glomerular Epithelium 0/3 0                                                   Mesangial 0/3 0                                                               Porximal Convoluted Tubules 3/3 500                                         Liver                                                                           Bile Duct           3/3       500                                             Central Lobular Hepatocyte 1/3 4                                              Periportal Hepatocyte 1/3 40                                                  Kupffer Cells 0/3 0                                                         Lung                                                                            Alveolar Macrophage 0/3       0                                               Bronchial Epithelium 0/2 0                                                    Bronchial Smooth Muscle 0/2 0                                                 Pneumocyte Type I 3/3 354                                                     Pneumocyte Type II 3/3 387                                                  Lymph Node                                                                      Lymphoid Follicle-Central                                                                         0/3       0                                               Lymphoid Follicle-Peripheral 0/3 0                                          Mammary Gland                                                                   Aveolar Epithelium  3/3       500                                             Duct Epithelium 3/3 500                                                       Mycepithelium 0/3 0                                                         Muscle Skeletal                                                                 Muscle Fiber        0/3       0                                             Nerve                                                                           Axon, Myelinated    0/2       0                                               Endoneurium 0/2 0                                                             Neurolemma 0/2 0                                                              Neuron 0/2 0                                                                  Perineurium 0/2 0                                                           Stomach                                                                         Chief Cells         3/3       290                                             Mucosal Epithelium 3/3 367                                                    Muscularis Mucosa/Externa 0/3 0                                             Stromal Tissue                                                                  Adipose              0/63     0                                               Arteriolar Smooth Muscle  0/120 0                                             Endothelium  0/120 0                                                          Fibrous Connective Tissue  0/120 0                                            Macrophages  0/117 0                                                          Mast Cells/Eosinophils  0/86 0                                              Testis                                                                          Interstitial Cells  0/2       0                                               Sertoli Cells 3/3 93                                                        Thymus                                                                          Hassal's Epithelium 3/3       147                                             Hassal's Keratin 3/3 333                                                      Lymphoid Cortex 0/3 0                                                         Lymphoid Medulla 3/3 167                                                    Thyroid                                                                         C-cells             0/3       0                                               Colloid 0/3 0                                                                 Follicular Epithelium 3/3 500                                               Tonsil                                                                          Epithelium          1/3       500                                             Lymphoid Follicle-Central 0/3 0                                               Lymphoid Follicle-Peripheral 0/3 0                                            Mucus Gland 1/1 300                                                           Striated Muscle 0/3 0                                                       Umbilical cord                                                                  Epiethlium          0/3       0                                             Urinary Bladder                                                                 Mucosal Epithelium  3/3       433                                             Serosa 0/1 0                                                                  Smooth Muscle 0/3 0                                                         Uterus                                                                          Endometrial Epithelium                                                                            3/3       500                                             Endometrial Glands 3/3 500                                                    Smooth Muscle 0/3 0                                                         Vagina/Cervix                                                                   Epithelial Glands   1/1       500                                             Smooth Muscle 0/2 0                                                           Squamous Epithelium 1/1 200                                                 ______________________________________                                    

The invention is further described through presentation of the followingexamples. These examples are offered by way of illustration, and not byway of limitation.

EXAMPLE I Synthesis of a Chelate-Biotin Conjugate

A chelating compound that contains an N₃ S chelating core was attachedvia an amide linkage to biotin. Radiometal labeling of an exemplarychelate-biotin conjugate is illustrated below. ##STR1##

The spacer group "X" permits the biotin portion of the conjugate to besterically available for avidin binding. When "R¹ " is a carboxylic acidsubstituent (for instance, CH₂ COOH), the conjugate exhibits improvedwater solubility, and further directs in vivo excretion of theradiolabeled biotin conjugate toward renal rather than hepatobiliaryclearance.

Briefly, N-α-Cbz-N-Σ-t-BOC protected lysine was converted to thesuccinimidyl ester with NHS and DCC, and then condensed with asparticacid β--t-butyl ester. The resultant dipeptide was activated with NHSand DCC, and then condensed with glycine t-butyl ester. The Cbz groupwas removed by hydrogenolysis, and the amine was acylated usingtetrahydropyranyl mercaptoacetic acid succinimidyl ester, yieldingS-(tetrahydropyranyl)-mercaptoacetyl-lysine. Trifluoroacetic acidcleavage of the N-t-BOC group and t-butyl esters, followed bycondensation with LC-biotin-NHS ester provided (Σ-caproylamidebiotin)-aspartyl glycine. This synthetic method is illustrated below.##STR2##

¹ H NMR: (CD₃ OD, 200 MHz Varian): 1.25-1.95 (m, 24H), 2.15-2.25 (broadt, 4H), 2.65-3.05 (m, 4H), 3.30-3.45 (dd, 2H), 3.50-3.65 (ddd, 2H), 3.95(broad s, 2H), 4.00-4.15 (m, 1H), 4.25-4.35 (m, 1H), 4.45-4.55 (m, 1H),4.7-5.05 (m overlapping with HOD).

Elemental Analysis: C, H, N for C₃₅ H₅₇ N₇ O₁₁ S₂. H₂ O; calculated:50.41, 7.13, 11.76; found: 50.13, 7.14, 11.40

EXAMPLE II Preparation of a Technetium or Rhenium RadiolabeledChelate-Biotin Conjugate

The chelate-biotin conjugate of Example I was radiolabeled with either^(99m) Tc pertechnetate or ¹⁸⁶ Re perrhenate. Briefly, ^(99m) Tcpertechnetate was reduced with stannous chloride in the presence ofsodium gluconate to form an intermediate Tc-gluconate complex. Thechelate-biotin conjugate of Example I was added and heated to 100° C.for 10 min at a pH of about 1.8 to about 3.3. The solution wasneutralized to a pH of about 6 to about 8, and yielded an N₃S-coordinated ^(99m) Tc-chelate-biotin conjugate. C-18 HPLC gradientelution using 5-60% acetonitrile in 1% acetic acid demonstrated twoanomers at 97% or greater radiochemical yield using δ (gamma ray)detection.

Alternatively, ¹⁸⁶ Re perrhenate was spiked with cold ammoniumperrhenate, reduced with stannous chloride, and complexed with citrate.The chelate-biotin conjugate of Example I was added and heated to 90° C.for 30 min at a pH of about 2 to 3. The solution was neutralized to a pHof about 6 to about 8, and yielded an N₃ S-coordinated ¹⁸⁶Re-chelate-biotin conjugate. C-18 HPLC gradient elution using 5-60%acetonitrile in 1% acetic acid resulted in radiochemical yields of85-90%. Subsequent purification over a C-18 reverse phase hydrophobiccolumn yielded material of 99% purity.

EXAMPLE III In Vitro Analysis of Radiolabeled Chelate-Biotin Conjugates

Both the ^(99m) Tc- and ¹⁸⁶ Re-chelate-biotin conjugates were evaluatedin vitro. When combined with excess avidin (about 100-fold molarexcess), 100% of both radiolabeled biotin conjugates complexed withavidin.

A ^(99m) Tc-biotin conjugate was subjected to various chemical challengeconditions. Briefly, ^(99m) Tc-chelate-biotin conjugates were combinedwith avidin and passed over a 5 cm size exclusion gel filtration column.The radiolabeled biotin-avidin complexes were subjected to variouschemical challenges (see Table 1), and the incubation mixtures werecentrifuged through a size exclusion filter. The percent ofradioactivity retained (indicating avidin-biotin-associated radiolabel)is presented in Table 1. Thus, upon chemical challenge, the radiometalremained associated with the macromolecular complex.

                  TABLE 1                                                         ______________________________________                                        Chemical Challenge of .sup.99m Tc-Chelate-                                      Biotin-Avidin Complexes                                                         Challenge             % Radioactivity Retained                            Medium      pH        1 h, 37° C.                                                                      18 h, RT                                      ______________________________________                                        PBS         7.2       99        99                                              Phosphate 8.0 97 97                                                           10 mM cysteine 8.0 92 95                                                      10 mM DTPA 8.0 99 98                                                          0.2M carbonate 10.0 97 94                                                   ______________________________________                                    

In addition, each radiolabeled biotin conjugate was incubated at about50 μg/ml with serum; upon completion of the incubation, the samples weresubjected to instant thin layer chromatography (ITLC) in 80% methanol.Only 2-4% of the radioactivity remained at the origin (i.e., associatedwith protein); this percentage was unaffected by the addition ofexogenous biotin. When the samples were analyzed using size exclusionH-12 FPLC with 0.2 M phosphate as mobile phase, no association ofradioactivity with serum macromolecules was observed.

Each radiolabeled biotin conjugate was further examined using acompetitive biotin binding assay. Briefly, solutions containing varyingratios of D-biotin to radiolabeled biotin conjugate were combined withlimiting avidin at a constant total biotin:avidin ratio. Avidin bindingof each radiolabeled biotin conjugate was determined by ITLC, and wascompared to the theoretical maximum stoichiometric binding (asdetermined by the HABA spectrophotometric assay of Green, Biochem. J.94:23c-24c, 1965). No significant difference in avidin binding wasobserved between each radiolabeled biotin conjugate and D-biotin.

EXAMPLE IV In Vivo Analysis of Radiolabeled Chelate-Biotin ConjugatesAdministered after Antibody Pretargeting

The ¹⁸⁶ Re-chelate-biotin conjugate of Example I was studied in ananimal model of a three-step antibody pretargeting protocol. Generally,this protocol involved: (i) prelocalization of biotinylated monoclonalantibody; (ii) administration of avidin for formation of a "sandwich" atthe target site and for clearance of residual circulating biotinylatedantibody; and (iii) administration of the 186Re-biotin conjugate fortarget site localization and rapid blood clearance.

A. Preparation and Characterization of Biotinylated Antibody

Biotinylated NR-LU-10 was prepared according to either of the followingprocedures. The first procedure involved derivitization of antibody vialysine ε-amino groups. NR-LU-10 was radioiodinated at tyrosines usingchloramine T and either ¹²⁵ I or ¹³¹ I sodium iodide. The radioiodinatedantibody (5-10 mg/ml) was then biotinylated using biotinamido caproateNHS ester in carbonate buffer, pH 8.5, containing 5% DMSO, according tothe scheme below. ##STR3##

The impact of lysine biotinylation on antibody immunoreactivity wasexamined. As the molar offering of biotin:antibody increased from 5:1 to40:1, biotin incorporation increased as expected (measured using theHABA assay and pronase-digested product) (Table 2, below). Percent ofbiotinylated antibody immunoreactivity as compared to native antibodywas assessed in a limiting antigen ELISA assay. The immunoreactivitypercentage dropped below 70% at a measured derivitization of 11.1:1;however, at this level of derivitization, no decrease was observed inantigen-positive cell binding (performed with LS-180 tumor cells atantigen excess). Subsequent experiments used antibody derivatized at abiotin:antibody ratio of 10:1.

                  TABLE 2                                                         ______________________________________                                        Effect of Lysine Biotinylation                                                  on Immunoreactivity                                                             Molar     Measured                                                          Offering Derivitization Immunoassessment (%)                                (Biotins/Ab)                                                                            (Biotins/Ab)  ELISA   Cell Binding                                  ______________________________________                                         5:1      3.4           86                                                      10:1 8.5 73 100                                                               13:1 11.1 69 102                                                              20:1 13.4 36 106                                                              40:1 23.1 27                                                                ______________________________________                                    

Alternatively, NR-LU-10 was biotinylated using thiol groups generated byreduction of cystines. Derivitization of thiol groups was hypothesizedto be less compromising to antibody immunoreactivity. NR-LU-10 wasradioiodinated using p-aryltin phenylate NHS ester (PIP-NHS) and either¹²⁵ I or ¹³¹ I sodium iodide. Radioiodinated NR-LU-10 was incubated with25 mM dithiothreitol and purified using size exclusion chromatography.The reduced antibody (containing free thiol groups) was then reactedwith a 10- to 100-fold molar excess of N-iodoacetyl-n'-biotinyl hexylenediamine in phosphate-buffered saline (PBS), pH 7.5, containing 5% DMSO(v/v).

                  TABLE 3                                                         ______________________________________                                        Effect of Thiol Biotinylation                                                   on Immunoreactivity                                                             Molar     Measured                                                          Offering Derivitization Immunoassessment (%)                                (Biotins/Ab)                                                                            (Biotins/Ab)  ELISA   Cell Binding                                  ______________________________________                                        10:1      4.7           114                                                     50:1 6.5 102 100                                                              100:1  6.1 95 100                                                           ______________________________________                                    

As shown in Table 3, at a 50:1 or greater biotin:antibody molaroffering, only 6 biotins per antibody were incorporated. No significantimpact on immunoreactivity was observed.

The lysine- and thiol-derivatized biotinylated antibodies ("antibody(lysine)" and "antibody (thiol)", respectively) were compared. Molecularsizing on size exclusion FPLC demonstrated that both biotinylationprotocols yielded monomolecular (monomeric) IgGs. Biotinylated antibody(lysine) had an apparent molecular weight of 160 kD, while biotinylatedantibody (thiol) had an apparent molecular weight of 180 kD. Reductionof endogenous sulfhydryls (i.e., disulfides) to thiol groups, followedby conjugation with biotin, may produce a somewhat unfoldedmacromolecule. If so, the antibody (thiol) may display a largerhydrodynamic radius and exhibit an apparent increase in molecular weightby chromatographic analysis. Both biotinylated antibody speciesexhibited 98% specific binding to immobilized avidin-agarose.

Further comparison of the biotinylated antibody species was performedusing non-reducing SDS-PAGE, using a 4% stacking gel and a 5% resolvinggel. Biotinylated samples were either radiolabeled or unlabeled and werecombined with either radiolabeled or unlabeled avidin or streptavidin.Samples were not boiled prior to SDS-PAGE analysis. The native antibodyand biotinylated antibody (lysine) showed similar migrations; thebiotinylated antibody (thiol) produced two species in the 50-75 kDrange. These species may represent two thiol-capped species. Under theseSDS-PAGE conditions, radiolabeled streptavidin migrates as a 60 kDtetramer. When 400 μg/ml radiolabeled streptavidin was combined with 50μg/ml biotinylated antibody (analogous to "sandwiching" conditions invivo), both antibody species formed large molecular weight complexes.However, only the biotinylated antibody (thiol)-streptavidin complexmoved from the stacking gel into the resolving gel, indicating adecreased molecular weight as compared to the biotinylated antibody(lysine)-streptavidin complex.

B. Blood Clearance of Biotinylated Antibody Species

Radioiodinated biotinylated NR-LU-10 (lysine or thiol) was intravenouslyadministered to non-tumored nude mice at a dose of 100 μg. At 24 hpost-administration of radioiodinated biotinylated NR-LU-10, mice wereintravenously injected with either saline or 400 μg of avidin. Withsaline administration, blood clearances for both biotinylated antibodyspecies were biphasic and similar to the clearance of native NR-LU-10antibody.

In the animals that received avidin intravenously at 24 h, thebiotinylated antibody (lysine) was cleared (to a level of 5% of injecteddose) within 15 min of avidin administration (avidin:biotin=10:1). Withthe biotinylated antibody (thiol), avidin administration (10:1 or 25:1)reduced the circulating antibody level to about 35% of injected doseafter two hours. Residual radiolabeled antibody activity in thecirculation after avidin administration was examined in vitro usingimmobilized biotin. This analysis revealed that 85% of the biotinylatedantibody was complexed with avidin. These data suggest that thebiotinylated antibody (thiol)-avidin complexes that were formed wereinsufficiently crosslinked to be cleared by the RES.

Blood clearance and biodistribution studies of biotinylated antibody(lysine) 2 h post-avidin or post-saline administration were performed.Avidin administration significantly reduced the level of biotinylatedantibody in the blood (see FIG. 1), and increased the level ofbiotinylated antibody in the liver and spleen. Kidney levels ofbiotinylated antibody were similar.

EXAMPLE V In Vivo Characterization of ¹⁸⁶ Re-Chelate-Biotin Conjugatesin a Three-Step Pretargeting Protocol

A ¹⁸⁶ Re-chelate-biotin conjugate of Example I (MW≈1000; specificactivity=1-2 mCi/mg) was examined in a three-step pretargeting protocolin an animal model. More specifically, 18-22 g female nude mice wereimplanted subcutaneously with LS-180 human colon tumor xenografts,yielding 100-200 mg tumors within 10 days of implantation.

NR-LU-10 antibody (MW≈150 kD) was radiolabeled with ¹²⁵ I/Chloramine Tand biotinylated via lysine residues-(as described in Example IV.A,above). Avidin (MW≈66 kD) was radiolabeled with ¹³¹ I/PIP-NHS (asdescribed for radioiodination of NR-LU-10 in Example IV.A., above). Theexperimental protocol was as follows:

    ______________________________________                                        Group 1:    Time 0, inject 100 μg .sup.125 I-labeled,                         biotinylated NR-LU-10                                                         Time 24 h, inject 400 μg .sup.131 I-labeled                                avidin                                                                        Time 26 h, inject 60 μg .sup.186 Re-chelate-                               biotin conjugate                                                             Group 2: Time 0, inject 400 μg .sup.131 I-labeled avidin                   (control) Time 2 h, inject 60 μg .sup.186 Re-chelate-                       biotin conjugate                                                             Group 3: Time 0, inject 60 μg .sup.186 Re-chelate-                         (control) biotin conjugate                                                  ______________________________________                                    

The three radiolabels employed in this protocol are capable of detectionin the presence of each other. It is also noteworthy that the sizes ofthe three elements involved are logarithmicallydifferent--antibody≈150,000; avidin≈66,000; and biotin 1,000.Biodistribution analyses were performed at 2, 6, 24, 72 and 120 h afteradministration of the ¹⁸⁶ Re-chelate-biotin conjugate.

Certain preliminary studies were performed in the animal model prior toanalyzing the ¹⁸⁶ Re-chelate-biotin conjugate in a three-steppretargeting protocol. First, the effect of biotinylated antibody onblood clearance of avidin was examined. These experiments showed thatthe rate and extent of avidin clearance was similar in the presence orabsence of biotinylated antibody. Second, the effect of biotinylatedantibody and avidin on blood clearance of the ¹⁸⁶ Re-chelate-biotinconjugate was examined; blood clearance was similar in the presence orabsence of biotinylated antibody and avidin. Further, antibodyimmunoreactivity was found to be uncompromised by biotinylation at thelevel tested.

Third, tumor uptake of biotinylated antibody administered at time 0 orof avidin administered at time 24 h was examined. The results of thisexperimentation are shown in FIG. 1. At 25 h, about 350 pmol/gbiotinylated antibody was present at the tumor; at 32 h the level wasabout 300 pmol/g; at 48 h, about 200 pmol/g; and at 120 h, about 100pmol/g. Avidin uptake at the same time points was about 250, 150, 50 and0 pmol/g, respectively. From the same experiment, tumor to blood ratioswere determined for biotinylated antibody and for avidin. From 32 h to120 h, the ratios of tumor to blood were very similar.

Rapid and efficient removal of biotinylated antibody from the blood bycomplexation with avidin was observed. Within two hours of avidinadministration, a 10-fold reduction in blood pool antibody concentrationwas noted (FIG. 1), resulting in a sharp increase in tumor to bloodratios. Avidin is cleared rapidly, with greater than 90% of the injecteddose cleared from the blood within 1 hour after administration. TheRe-186-biotin chelate is also very rapidly cleared, with greater than99% of the injected dose cleared from the blood by 1 hour afteradministration.

The three-step pretargeting protocol (described for Group 1, above) wasthen examined. More specifically, tumor uptake of the ¹⁸⁶Re-chelate-biotin conjugate in the presence or absence of biotinylatedantibody and avidin was determined. In the absence of biotinylatedantibody and avidin, the ¹⁸⁶ Re-chelate-biotin conjugate displayed aslight peak 2 h post-injection, which was substantially cleared from thetumor by about 5 h. In contrast, at 2 h post-injection in the presenceof biotinylated antibody and avidin (specific), the ¹⁸⁶Re-chelate-biotin conjugate reached a peak in tumor approximately 7times greater than that observed in the absence of biotinylated antibodyand avidin. Further, the specifically bound ¹⁸⁶ Re-chelate-biotinconjugate was retained at the tumor at significant levels for more than50 h. Tumor to blood ratios determined in the same experiment increasedsignificantly over time (i.e., T:B=≈8 at 30 h; ≈15 at 100 h; ≈35 at 140h).

Tumor uptake of the ¹⁸⁶ Re-chelate-biotin conjugate has further beenshown to be dependent on the dose of biotinylated antibody administered.At 0 μg of biotinylated antibody, about 200 pmol/g of ¹⁸⁶Re-chelate-biotion conjugate was present at the tumor at 2 h afteradministration; at 50 μg antibody, about 500 pmol/g of186Re-chelate-biotin conjugate; and at 100 μg antibody, about 1,300pmol/g of ¹⁸⁶ Re-chelate-biotin conjugate.

Rhenium tumor uptake via the three-step pretargeting protocol wascompared to tumor uptake of the same antibody radiolabeled throughchelate covalently attached to the antibody (conventional procedure).The results of this comparison are depicted in FIG. 2. Blood clearanceand tumor uptake were compared for the chelate directly labeled rheniumantibody conjugate and for the three-step pretargeted sandwich. Areasunder the curves (AUC) and the ratio of AUC_(tumor) /AUC_(blood) weredetermined. For the chelate directly labeled rhenium antibody conjugate,the ratio of AUC_(tumor) /AUC_(blood) =24055/10235 or 2.35; for thethree-step pretargeted sandwich, the ratio of AUC_(tumor) /AUC_(blood)=46764/6555 or 7.13.

Tumor uptake results are best taken in context with radioactivityexposure to the blood compartment, which directly correlates with bonemarrow exposure. Despite the fact that 100-fold more rhenium wasadministered to animals in the three-step protocol, the very rapidclearance of the small molecule (Re-186-biotin) from the blood minimizesthe exposure to Re-186 given in this manner. In the same matchedantibody dose format, direct labeled (conventional procedure) NR-LU-10whole antibody yielded greater exposure to rhenium than did the 100-foldhigher dose given in the three-step protocol. A clear increase in thetargeting ratio (tumor exposure to radioactivity:blood exposure toradioactivity--AUC_(tumor) :AUC_(blood)) was observed for three-steppretargeting (approximately 7:1) in comparison to the direct labeledantibody approach (approximately 2.4:1).

EXAMPLE VI Preparation of Chelate-Biotin Conjugates having ImprovedBiodistribution Properties

The biodistribution of ¹¹¹ In-labeled-biotin derivatives varies greatlywith structural changes in the chelate and the conjugating group.Similar structural changes may affect the biodistribution of technetium-and rhenium-biotin conjugates. Accordingly, methods for preparingtechnetium- and rhenium-biotin conjugates having optimal clearance fromnormal tissue are advantageous.

A. Neutral MAMA Chelate/Conjugate

A neutral MAMA chelate-biotin conjugate is prepared according to thefollowing scheme. ##STR4## The resultant chelate-biotin conjugate showssuperior kidney excretion. Although the net overall charge of theconjugate is neutral, the polycarboxylate nature of the moleculegenerates regions of hydrophilicity and hydrophobicity. By altering thenumber and nature of the carboxylate groups within the conjugate,excretion may be shifted from kidney to gastrointestinal routes. Forinstance, neutral compounds are generally cleared by the kidneys;anionic compounds are generally cleared through the GI system.

B. Polylysine Derivitization

Conjugates containing polylysine may also exhibit beneficialbiodistribution properties. With whole antibodies, derivitization withpolylysine may skew the biodistribution of conjugate toward liveruptake. In contrast, derivitization of Fab fragments with polylysineresults in lower levels of both liver and kidney uptake; blood clearanceof these conjugates is similar to that of Fab covalently linked tochelate. An exemplary polylysine derivatized chelate-biotin conjugate isillustrated below. ##STR5## Inclusion of polylysine inradiometal-chelate-biotin conjugates is therefore useful for minimizingor eliminating RES sequestration while maintaining good liver and kidneyclearance of the conjugate. For improved renal excretion properties,polylysine derivatives are preferably succinylated followingbiotinylation. Polylysine derivatives offer the further advantages of:(1) increasing the specific activity of the radiometal-chelate-biotinconjugate; (2) permitting control of rate and route of blood clearanceby varying the molecular weight of the polylysine polymer; and (3)increasing the circulation half-life of the conjugate for optimal tumorinteraction.

Polylysine derivitization is accomplished by standard methodologies.Briefly, poly-L-lysine is acylated according to standard amino groupacylation procedures (aqueous bicarbonate buffer, pH 8, added biotin-NHSester, followed by chelate NHS ester). Alternative methodology involvesanhydrous conditions using nitrophenyl esters in DMSO and triethylamine. The resultant conjugates are characterized by UV and NMR spectra.

The number of biotins attached to polylysine is determined by the HABAassay. Spectrophotometric titration is used to assess the extent ofamino group derivitization. The radiometal-chelate-biotin conjugate ischaracterized by size exclusion.

C. Cleavable Linkage

Through insertion of a cleavable linker between the chelate and biotinportion of a radiometal-chelate-biotin conjugate, retention of theconjugate at the tumor relative to normal tissue may be enhanced. Morespecifically, linkers that are cleaved by enzymes present in normaltissue but deficient or absent in tumor tissue can increase tumorretention. As an example, the kidney has high levels of γ-glutamyltransferase; other normal tissues exhibit in vivo cleavage of γ-glutamylprodrugs. In contrast, tumors are generally deficient in enzymepeptidases. The glutamyl-linked biotin conjugate depicted below iscleaved in normal tissue and retained in the tumor. ##STR6## D. SerineLinker with O-Polar Substituent

Sugar substitution of N₃ S chelates renders such chelates water soluble.Sulfonates, which are fully ionized at physiological pH, improve watersolubility of the chelate-biotin conjugate depicted below. ##STR7## Thiscompound is synthesized according to the standard reaction procedures.Briefly, biocytin is condensed with N-t-BOC-(O-sulfonate or O-glucose)serine NHS ester to give N-t-BOC-(O-sulfonate or O-glucose) serinebiocytinamide. Subsequent cleavage of the N-t-BOC group with TFA andcondensation with ligand NHS ester in DMF with triethylamine providesligand-amidoserine(O-sulfonate or O-glucose)biocytinamide.

EXAMPLE VII Preparation and Characterization of PIP-RadioiodinatedBiotin

Radioiodinated biotin derivatives prepared by exposure of poly-L-lysineto excess NHS-LC-biotin and then to Bolton-Hunter N-hydroxysuccinimideesters in DMSO has been reported. After purification, this product wasradiolabeled by the iodogen method (see, for instance, Del Rosario etal., J. Nucl. Med. 32:5, 1991, 993 (abstr.)). Because of the highmolecular weight of the resultant radioiodinated biotin derivative, onlylimited characterization of product (i.e., radio-HPLC and binding toimmobilized streptavidin) was possible.

Preparation of radioiodinated biotin according to the present inventionprovides certain advantages. First, the radioiodobiotin derivative is alow molecular weight compound that is amenable to complete chemicalcharacterization. Second, the disclosed methods for preparation involvea single step and eliminate the need for a purification step.

Briefly, iodobenzamide derivatives corresponding to biocytin (R=COOH)and biotinamidopentylamine (R=H) were prepared according to thefollowing scheme. In this scheme, "X" may be any radiohalogen, including¹²⁵ I, ¹³¹ I, ¹²³ I, ²¹¹ At and the like. ##STR8## Preparation of 1 wasgenerally according to Wilbur et al., J. Nucl. Med. 30:216-26, 1989,using a tributyltin intermediate. Water soluble carbodiimide was used inthe above-depicted reaction, since the NHS ester 1 formed intractablemixtures with DCU. The NHS ester was not compatible with chromatography;it was insoluble in organic and aqueous solvents and did not react withbiocytin in DMF or in buffered aqueous acetonitrile. The reactionbetween 1 and biocytin or 5-(biotinamido) pentylamine was sensitive tobase. When the reaction of 1 and biocytin or the pentylamine wasperformed in the presence of triethylamine in hot DMSO, formation ofmore than one biotinylated product resulted. In contrast, the reactionwas extremely clean and complete when a suspension of 1 and biocytin (4mg/ml) or the pentylamine (4 mg/ml) was heated in DMSO at 117° C. forabout 5 to about 10 min. The resultant ¹²⁵ I-biotin derivatives wereobtained in 94% radiochemical yield. Optionally, the radioiodinatedproducts may be purified using C-18 HPLC and a reverse phase hydrophobiccolumn. Hereinafter, the resultant radioiodinated products 2 arereferred to as PIP-biocytin (R=COOH) and PIP-pentylamine (R=H).

Both iodobiotin derivatives 2 exhibited ≧95% binding to immobilizedavidin. Incubation of the products 2 with mouse serum resulted in noloss of the ability of 2 to bind to immobilized avidin. Biodistributionstudies of 2 in male BALB/c mice showed rapid clearance from the blood(similar to ¹⁸⁶ Re-chelate-biotin conjugates described above). Theradioiodobiotin 2 had decreased hepatobiliary excretion as compared tothe ¹⁸⁶ Re-chelate-biotin conjugate; urinary excretion was increased ascompared to the ¹⁸⁶ Re-chelate-biotin conjugate. Analysis of urinarymetabolites of 2 indicated deiodination and cleavage of the biotin amidebond; the metabolites showed no binding to immobilized avidin. Incontrast, metabolites of the ¹⁸⁶ Re-chelate-biotin conjugate appear tobe excreted in urine as intact biotin conjugates. Intestinal uptake of 2is <50% that of the ¹⁸⁶ Re-chelate-biotin conjugate. Thesebiodistribution properties of 2 provided enhanced whole body clearanceof radioisotope and indicate the advantageous use of 2 withinpretargeting protocols.

¹³¹ I-PIP-biocytin was evaluated in a two-step pretargeting procedure intumor-bearing mice. Briefly, female nude mice were injectedsubcutaneously with LS-180 tumor cells; after 7 d, the mice displayed50-100 mg tumor xenografts. At t=0, the mice were injected with 200 μgof NR-LU-10-streptavidin conjugate labeled with ¹²⁵.sub. I using PIP-NHS(see Example IV.A.). At t=36 h, the mice received 42 μg of ¹³¹IPIP-biocytin. The data showed immediate, specific tumor localization,corresponding to ≈1.5 ¹³¹ I-PIP-biocytin molecules per avidin molecule.

The described radiohalogenated biotin compounds are amenable to the sametypes of modifications described in Example VI above for ¹⁸⁶Re-chelate-biotin conjugates. In particular, the followingPIP-polylysine-biotin molecule is made by trace labeling polylysine with¹²⁵ I-PIP, followed by extensive biotinylation of the polylysine.##STR9## Assessment of ¹²⁵ I binding to immobilized avidin ensures thatall radioiodinated species also contain at least an equivalent ofbiotin.

EXAMPLE VIII Preparation of Biotinylated Antibody (Thiol) ThroughEndogenous Antibody Sulfhydryl Groups or Sulfhydryl-Generating Compounds

Certain antibodies have available for reaction endogenous sulfhydrylgroups. If the antibody to be biotinylated contains endogenoussulfhydryl groups, such antibody is reacted withN-iodoacetyl-n'-biotinyl hexylene diamine (as described in ExampleIV.A., above). The availability of one or more endogenous sulfhydrylgroups obviates the need to expose the antibody to a reducing agent,such as DTT, which can have other detrimental effects on thebiotinylated antibody.

Alternatively, one or more sulfhydryl groups are attached to a targetingmoiety through the use of chemical compounds or linkers that contain aterminal sulfhydryl group. An exemplary compound for this purpose isiminothiolane. As with endogenous sulfhydryl groups (discussed above),the detrimental effects of reducing agents on antibody are therebyavoided.

EXAMPLE IX Two-Step Pretargeting Methodology that does not InduceInternalization

A NR-LU-13-avidin conjugate is prepared as follows. Initially, avidin isderivatized with N-succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC). SMCC-derivedavidin is then incubated with NR-LU-13 in a 1:1 molar ratio at pH 8.5for 16 h. Unreacted NR-LU-13 and SMCC-derived avidin are removed fromthe mixture using preparative size exclusion HPLC. Two conjugates areobtained as products--the desired 1:1 NR-LU-13-avidin conjugate as themajor product; and an incompletely characterized component as the minorproduct.

A ^(99m) Tc-chelate-biotin conjugate is prepared as in Example II,above. The NR-LU-13-avidin conjugate is administered to a recipient andallowed to clear from the circulation. One of ordinary skill in the artof radioimmunoscintigraphy is readily able to determine the optimal timefor NR-LU-13-avidin conjugate tumor localization and clearance from thecirculation. At such time, the ^(99m) Tc-chelate-biotin conjugate isadministered to the recipient. Because the ^(99m) Tc-chelate-biotinconjugate has a molecular weight of ≈1,000, crosslinking ofNR-LU-13-avidin molecules on the surface of the tumor cells isdramatically reduced or eliminated. As a result, the ^(99m) Tcdiagnostic agent is retained at the tumor cell surface for an extendedperiod of time. Accordingly, detection of the diagnostic agent byimaging techniques is optimized; further, a lower dose of radioisotopeprovides an image comparable to that resulting from the typicalthree-step pretargeting protocol.

Optionally, clearance of NR-LU-13-avidin from the circulation may beaccelerated by plasmapheresis in combination with a biotin affinitycolumn. Through use of such column, circulating NR-LU-13-avidin will beretained extracorporeally, and the recipient's immune system exposure toa large, proteinaceous immunogen (i.e., avidin) is minimized.

Exemplary methodology for plasmapheresis/column purification useful inthe practice of the present invention is discussed in the context ofreducing radiolabeled antibody titer in imaging and in treating tumortarget sites in U.S. Pat. No. 5,078,673. Briefly, for the purposes ofthe present invention, an example of an extracorporeal clearancemethodology may include the following steps:

administering a ligand- or anti-ligand-targeting moiety conjugate to arecipient;

after a time sufficient for localization of the administered conjugateto the target site, withdrawing blood from the recipient by, forexample, plasmapheresis;

separating cellular element from said blood to produce a serum fractionand returning the cellular elements to the recipient; and

reducing the titer of the administered conjugate in the serum fractionto produce purified serum;

infusing the purified serum back into the recipient.

Clearance of NR-LU-13-avidin is also facilitated by administration of aparticulate-type clearing agent (e.g., a polymeric particle having aplurality of biotin molecules bound thereto). Such a particulateclearing agent preferably constitutes a biodegradable polymeric carrierhaving a plurality of biotin molecules bound thereto. Particulateclearing agents of the present invention exhibit the capability ofbinding to circulating administered conjugate and removing thatconjugate from the recipient. Particulate clearing agents of this aspectof the present invention may be of any configuration suitable for thispurpose. Preferred particulate clearing agents exhibit one or more ofthe following characteristics:

microparticulate (e.g., from about 0.5 micrometers to about 100micrometers in diameter, with from about 0.5 to about 2 micrometers morepreferred), free flowing powder structure;

biodegradable structure designed to biodegrade over a period of timebetween from about 3 to about 180 days, with from about 10 to about 21days more preferred, or non-biodegradable structure;

biocompatible with the recipients physiology over the course ofdistribution, metabolism and excretion of the clearing agent, morepreferably including biocompatible biodegradation products;

and capability to bind with one or more circulating conjugates tofacilitate the elimination or removal thereof from the recipient throughone or more binding moieties (preferably, the complementary member ofthe ligand/anti-ligand pair). The total molar binding capacity of theparticulate clearing agents depends upon the particle size selected andthe ligand or anti-ligand substitution ratio. The binding moieties arecapable of coupling to the surface structure of the particulate dosageform through covalent or non-covalent modalities as set forth herein toprovide accessible ligand or anti-ligand for binding to its previouslyadministered circulating binding pair member.

Preferable particulate clearing agents of the present invention arebiodegradable or non-biodegradable microparticulates. More preferably,the particulate clearing agents are formed of a polymer containingmatrix that biodegrades by random, nonenzymatic, hydrolytic scissioning.

Polymers derived from the condensation of alpha hydroxycarboxylic acidsand related lactones are more preferred for use in the presentinvention. A particularly preferred moiety is formed of a mixture ofthermoplastic polyesters (e.g., polylactide or polyglycolide) or acopolymer of lactide and glycolide components, such aspoly(lactide-co-glycolide). An exemplary structure, a randompoly(DL-lactide-co-glycolide), is shown below, with the values of x andy being manipulable by a practitioner in the art to achieve desirablemicroparticulate properties. ##STR10##

Other agents suitable for forming particulate clearing agents of thepresent invention include polyorthoesters and polyacetals (PolymerLetters, 18:293, 1980) and polyorthocarbonates (U.S. Pat. No. 4,093,709)and the like.

Preferred lactic acid/glycolic acid polymer containing matrixparticulates of the present invention are prepared by emulsion-basedprocesses, that constitute modified solvent extraction processes such asthose described by Cowsar et al., "Poly(Lactide-Co-Glycolide)Microcapsules for Controlled Release of Steroids," Methods Enzymology,112:101-116, 1985 (steroid entrapment in microparticulates); Eldridge etal., "Biodegradable and Biocompatible Poly(DL-Lactide-Co-Glycolide)Microspheres as an Adjuvant for Staphylococcal Enterotoxin B ToxoidWhich Enhances the Level of Toxin-Neutralizing Antibodies," Infectionand Immunity, 59:2978-2986, 1991 (toxoid entrapment); Cohen et al.,"Controlled Delivery Systems for Proteins Based on Poly(Lactic/GlycolicAcid) Microspheres," Pharmaceutical Research, 8(6):713-720, 1991 (enzymeentrapment); and Sanders et al., "Controlled Release of a LuteinizingHormone-Releasing Hormone Analogue from Poly(D,L-Lactide-Co-Glycolide)Microspheres," J. Pharmaceutical Science, 73(9):1294-1297, 1984 (peptideentrapment).

In general, the procedure for forming particulate clearing agents of thepresent invention involves dissolving the polymer in a halogenatedhydrocarbon solvent and adding an additional agent that acts as asolvent for the halogenated hydrocarbon solvent but not for the polymer.The polymer precipitates out from the polymer-halogenated hydrocarbonsolution. Following particulate formation, they are washed and hardenedwith an organic solvent. Water washing and aqueous non-ionic surfactantwashing steps follow, prior to drying at room temperature under vacuum.

For biocompatibility purposes, particulate clearing agents aresterilized prior to packaging, storage or administration. Sterilizationmay be conducted in any convenient manner therefor. For example, theparticulates can be irradiated with gamma radiation, provided thatexposure to such radiation does not adversely impact the structure orfunction of the binding moiety attached thereto. If the binding moietyis so adversely impacted, the particulate clearing agents can beproduced under sterile conditions.

The preferred lactide/glycolide structure is biocompatible with themammalian physiological environment. Also, these preferred sustainedrelease dosage forms have the advantage that biodegradation thereofforms lactic acid and glycolic acid, both normal metabolic products ofmammals.

Functional groups required for binding moiety--particulate bonding, areoptionally included in the particulate structure, along with thenon-degradable or biodegradable polymeric units. Functional groups thatare exploitable for this purpose include those that are reactive withligands or anti-ligands, such as carboxyl groups, amine groups,sulfhydryl groups and the like. Preferred binding enhancement moietiesinclude the terminal carboxyl groups of the preferred(lactide-glycolide) polymer containing matrix or the like. Apractitioner in the art is capable of selecting appropriate functionalgroups and monitoring conjugation reactions involving those functionalgroups.

Advantages garnered through the use of particulate clearing agents ofthe type described above are as follows:

particles in the "micron" size range localize in the RES and liver, withgalactose derivatization or charge modification enhancement methods forthis capability available, and, preferably, are designed to remain incirculation for a time sufficient to perform the clearance function;

the size of the particulates facilitates central vascular compartmentretention thereof, substantially precluding equilibration into theperipheral or extravascular compartment;

desired substituents for ligand or anti-ligand binding to theparticulates can be introduced into the polymeric structure;

ligand- or anti-ligand-particulate linkages having desired properties(e.g., serum biotinidase resistance thereby reducing the release ofbiotin metabolite from a particle-biotin clearing agent) and

multiple ligands or anti-ligands can be bound to the particles toachieve optimal cross-linking of circulating targeting agent-ligand or-anti-ligand conjugate and efficient clearance of cross-linked species.This advantage is best achieved when care is taken to preventparticulate aggregation both in storage and upon in vivo administration.

Clearance of NR-LU-13-avidin may also be accelerated by an arteriallyinserted proteinaceous or polymeric multiloop device. A catheter-likedevice, consisting of thin loops of synthetic polymer or protein fibersderivatized with biotin, is inserted into a major artery (e.g., femoralartery) to capture NR-LU-13-avidin. Since the total blood volume passesthrough a major artery every 70 seconds, the in situ clearing device iseffective to reduce circulating NR-LU-13-avidin within a short period oftime. This device offers the advantages that NR-LU-13-avidin is notprocessed through the RES; removal of NR-LU-13-avidin is controllableand measurable; and fresh devices with undiminished binding capacity areinsertable as necessary. This methodology is also useful withintraarterial administration embodiments of the present invention.

An alternative procedure for clearing NR-LU-13-avidin from thecirculation without induction of internalization involves administrationof biotinylated, high molecular weight molecules, such as liposomes, IgMand other molecules that are size excluded from ready permeability totumor sites. When such biotinylated, high molecular weight moleculesaggregate with NR-LU-13-avidin, the aggregated complexes are readilycleared from the circulation via the RES.

EXAMPLE X Enhancement of Therapeutic Agent Internalization ThroughAvidin Crosslinking

The ability of multivalent avidin to crosslink two or more biotinmolecules (or chelate-biotin conjugates) is advantageously used toimprove delivery of therapeutic agents. More specifically, avidincrosslinking induces internalization of crosslinked complexes at thetarget cell surface.

Biotinylated NR-CO-04 (lysine) is prepared according to the methodsdescribed in Example IV.A., above. Doxorubicin-avidin conjugates areprepared by standard conjugation chemistry. The biotinylated NR-CO-04 isadministered to a recipient and allowed to clear from the circulation.One of ordinary skill in the art of radioimmunotherapy is readily ableto determine the optimal time for biotinylated NR-CO-04 tumorlocalization and clearance from the circulation. At such time, thedoxorubicin-avidin conjugate is administered to the recipient. Theavidin portion of the doxorubicin-avidin conjugate crosslinks thebiotinylated NR-CO-04 on the cell surface, inducing internalization ofthe complex. Thus, doxorubicin is more efficiently delivered to thetarget cell.

In a first alternative protocol, a standard three-step pretargetingmethodology is used to enhance intracellular delivery of a drug to atumor target cell. By analogy to the description above, biotinylatedNR-LU-05 is administered, followed by avidin (for blood clearance and toform the middle layer of the sandwich at the target cell-boundbiotinylated antibody). Shortly thereafter, and prior to internalizationof the biotinylated NR-LU-05-avidin complex, a methotrexate-biotinconjugate is administered.

In a second alternative protocol, biotinylated NR-LU-05 is furthercovalently linked to methotrexate. Subsequent administration of avidininduces internalization of the complex and enhances intracellulardelivery of drug to the tumor target cell.

In a third alternative protocol, NR-CO-04-avidin is administered to arecipient and allowed to clear from the circulation and localize at thetarget site. Thereafter, a polybiotinylated species (such asbiotinylated poly-L-lysine, as in Example IV.B., above) is administered.In this protocol, the drug to be delivered may be covalently attached toeither the antibody-avidin component or to the polybiotinylated species.The polybiotinylated species induces internalization of the(drug)-antibody-avidin-polybiotin-(drug) complex.

EXAMPLE XI Targeting Moiety-Anti-Ligand Conjugate for Two-StepPretargeting in Vivo

A. Preparation of SMCC-derivatized streptavidin.

31 mg (0.48 μmol) streptavidin was dissolved in 9.0 ml PBS to prepare afinal solution at 3.5 mg/ml. The pH of the solution was adjusted to 8.5by addition of 0.9 ml of 0.5 M borate buffer, pH 8.5. A DMSO solution ofSMCC (3.5 mg/ml) was prepared, and 477 μl (4.8 μmol) of this solutionwas added dropwise to the vortexing protein solution. After 30 minutesof stirring, the solution was purified by G-25 (PD-10, Pharmacia,Piscataway, N.J.) column chromatography to remove unreacted orhydrolyzed SMCC. The purified SMCC-derivatized streptavidin was isolated(28 mg, 1.67 mg/ml).

B. Preparation of DTT-reduced NR-LU-10. To 77 mg NR-LU-10 (0.42 μmol) in15.0 ml PBS was added 1.5 ml of 0.5 M borate buffer, pH 8.5. A DTTsolution, at 400 mg/ml (165 μl) was added to the protein solution. Afterstirring at room temperature for 30 minutes, the reduced antibody waspurified by G-25 size exclusion chromatography. Purified DTT-reducedNR-LU-10 was obtained (74 mg, 2.17 mg/ml).

C. Coniugation of SMCC-streptavidin to DTT-reduced NR-LU-10. DTT-reducedNR-LU-10 (63 mg, 29 ml, 0.42 μmol) was diluted with 44.5 ml PBS. Thesolution of SMCC-streptavidin (28 mg, 17 ml, 0.42 μmol) was addedrapidly to the stirring solution of NR-LU-10. Total proteinconcentration in the reaction mixture was 1.0 mg/ml. The progress of thereaction was monitored by HPLC (Zorbax® GF-250, available from MacMod).After approximately 45 minutes, the reaction was quenched by addingsolid sodium tetrathionate to a final concentration of 5 mM.

D. Purification of conjugate. For small scale reactions, monosubstitutedor disubstituted with regard to streptavidin conjugate was obtainedusing HPLC Zorbax (preparative) size exclusion chromatography. Thedesired monosubstituted or disubstituted conjugate product eluted at14.0-14.5 min (3.0 ml/min flow rate), while unreacted NR-LU-10 eluted at14.5-15 min and unreacted derivatized streptavidin eluted at 19-20 min.

For larger scale conjugation reactions, monosubstituted or disubstitutedadduct is isolatable using DEAE ion exchange chromatography. Afterconcentration of the crude conjugate mixture, free streptavidin wasremoved therefrom by eluting the column with 2.5% xylitol in sodiumborate buffer, pH 8.6. The bound unreacted antibody and desiredconjugate were then sequentially eluted from the column using anincreasing salt gradient in 20 mM diethanolamine adjusted to pH 8.6 withsodium hydroxide.

E. Characterization of Conjugate.

1. HPLC size exclusion was conducted as described above with respect tosmall scale purification.

2. SDS-PAGE analysis was performed using 5% polyacrylamide gels undernon-denaturing conditions. Conjugates to be evaluated were not boiled insample buffer containing SDS to avoid dissociation of streptavidin intoits 15 kD subunits. Two product bands were observed on the gel, whichcorrespond to the mono- and di-substituted conjugates.

3. Immunoreactivity was assessed, for example, by competitive bindingELISA as compared to free antibody. Values obtained were within 10% ofthose for the free antibody.

4. Biotin binding capacity was assessed, for example, by titrating aknown quantity of conjugate with p-[I-125]iodobenzoylbiocytin.Saturation of the biotin binding sites was observed upon addition of 4equivalences of the labeled biocytin.

5. In vivo studies are useful to characterize the reaction product,which studies include, for example, serum clearance profiles, ability ofthe conjugate to target antigen-positive tumors, tumor retention of theconjugate over time and the ability of a biotinylated molecule to bindstreptavidin conjugate at the tumor. These data facilitate determinationthat the synthesis resulted in the formation of a 1:1streptavidin-NR-LU-10 whole antibody conjugate that exhibits bloodclearance properties similar to native NR-LU-10 whole antibody, andtumor uptake and retention properties at least equal to native NR-LU-10.

For example, FIG. 3 depicts the tumor uptake profile of theNR-LU-10-streptavidin conjugate (LU-10-StrAv) in comparison to a controlprofile of native NR-LU-10 whole antibody. LU-10-StrAv was radiolabeledon the streptavidin component only, giving a clear indication thatLU-10-StrAv localizes to target cells as efficiently as NR-LU-10 wholeantibody itself.

EXAMPLE XII Two-Step Pretargeting in Vivo

A ¹⁸⁶ Re-chelate-biotin conjugate (Re-BT) of Example I (MW≈1000;specific activity=1-2 mCi/mg) and a biotin-iodine-131 small molecule,PIP-Biocytin (PIP-BT, MW approximately equal to 602; specific activity=0.5-1.0 mCi/mg), as discussed in Example VII above, were examined in athree-step pretargeting protocol in an animal model, as described inExample V above. Like Re-BT, PIP-BT has the ability to bind well toavidin and is rapidly cleared from the blood, with a serum half-life ofabout 5 minutes. Equivalent results were observed for both molecules inthe two-step pretargeting experiments described herein.

NR-LU-10 antibody (MW≈150 kD) was conjugated to streptavidin (MW≈66 kD)(as described in Example XI above) and radiolabeled with ¹²⁵ I/PIP-NHS(as described for radioiodination of NR-LU-10 in Example IV.A., above).The experimental protocol was as follows:

    ______________________________________                                        Time 0        inject (i.v.) 200 μg NR-LU-10-StrAv                             conjugate;                                                                   Time 24-48 h inject (i.v.) 60-70 fold                                          molar excess of                                                               radiolabeled biotinyl                                                         molecule;                                                                  and perform biodistributions at 2, 6, 24, 72, 120                               hours after injection of radiolabeled biotinyl                                molecule                                                                    ______________________________________                                    

NR-LU-10-streptavidin has shown very consistent patterns of bloodclearance and tumor uptake in the LS-180 animal model. A representativeprofile is shown in FIG. 4. When either PIP-BT or Re-BT is administeredafter allowing the LU-10-StrAv conjugate to localize to target cellsites for at least 24 hours, the tumor uptake of therapeuticradionuclide is high in both absolute amount and rapidity. For PIP-BTadministered at 37 hours following LU-10-StrAv (I-125) administration,tumor uptake was above 500 pMOL/G at the 40 hour time point and peakedat about 700 pMOL/G at 45 hours post-LU-10-StrAv administration.

This almost instantaneous uptake of a small molecule therapeutic intotumor in stoichiometric amounts comparable to the antibody targetingmoiety facilitates utilization of the therapeutic radionuclide at itshighest specific activity. Also, the rapid clearance of radionuclidethat is not bound to LU-10-StrAv conjugate permits an increasedtargeting ratio (tumor:blood) by eliminating the slow tumor accretionphase observed with directly labeled antibody conjugates. The pattern ofradionuclide tumor retention is that of whole antibody, which is verypersistent.

Experimentation using the two-step pretargeting approach andprogressively lower molar doses of radiolabeled biotinyl molecule wasalso conducted. Uptake values of about 20% ID/G were achieved atno-carrier added (high specific activity) doses of radiolabeled biotinylmolecules. At less than saturating doses, circulating LU-10-StrAv wasobserved to bind significant amounts of administered radiolabeledbiotinyl molecule in the blood compartment.

EXAMPLE XIII Asialoorosomucoid Clearing Agent and Two-Step Pretargeting

In order to maximize the targeting ratio (tumor:blood), clearing agentswere sought that are capable of clearing the blood pool of targetingmoiety-anti-ligand conjugate (e.g., LU-10-StrAv), without compromisingthe ligand binding capacity thereof at the target sites. One such agent,biotinylated asialoorosomucoid, which employs the avidin-biotininteraction to conjugate to circulating LU-10-StrAv, was tested.

A. Derivitization of orosomucoid. 10 mg human orosomucoid (Sigma N-9885)was dissolved in 3.5 ml of pH 5.5 0.1 M sodium acetate buffer containing160 mM NaCl. 70 μl of a 2% (w/v) CaCl solution in deionized (D.I.) waterwas added and 11 μl of neuraminidase (Sigma N-7885), 4.6 U/ml, wasadded. The mixture was incubated at 37° C. for 2 hours, and the entiresample was exchanged over a Centricon-10® ultrafiltration device(available from Amicon, Danvers, Mass.) with 2 volumes of PBS. Theasialoorosomucoid and orosomucoid starting material were radiolabeledwith I-125 using PIP technology, as described in Example IV above.

The two radiolabeled preparations were injected i.v. into female BALB/cmice (20-25 g), and blood clearance was assessed by serial retro-orbitaleye bleeding of each group of three mice at 5, 10, 15 and 30 minutes, aswell as at 1, 2 and 4 hours post-administration. The results of thisexperiment are shown in FIG. 5, with asialoorosomucoid clearing morerapidly than its orosomucoid counterpart.

In addition, two animals receiving each compound were sacrificed at 5minutes post-administration and limited biodistributions were performed.These results are shown in FIG. 6. The most striking aspects of thesedata are the differences in blood levels (78% for orosomucoid and 0.4%for asialoorosomucoid) and the specificity of uptake ofasialoorosomucoid in the liver (86%), as opposed to other tissues.

B. Biotinylation of asialoorosomucoid clearing agent and orosomucoidcontrol. 100 μl of 0.2 M sodium carbonate buffer, pH 9.2, was added to 2mg (in 1.00 ml PBS) of PIP-125-labeled orosomucoid and to 2 mgPIP-125-labeled asialoorosomucoid. 60 μl of a 1.85 mg/ml solution ofNHS-amino caproate biotin in DMSO was then added to each compound. Thereaction mixtures were vortexed and allowed to sit at room temperaturefor 45 minutes. The material was purified by size exclusion columnchromatography (PD-10, Pharmacia) and eluted with PBS. 1.2 ml fractionswere taken, with fractions 4 and 5 containing the majority of theapplied radioactivity (>95%). Streptavidin-agarose beads (Sigma S-1638)or -pellets were washed with PBS, and 20 μg of each biotinylated,radiolabeled protein was added to 400 μl of beads and 400 μl of PBS,vortexed for 20 seconds and centrifuged at 14,000 rpm for 5 minutes. Thesupernatant was removed and the pellets were washed with 400 μl PBS.This wash procedure was repeated twice more, and the combinedsupernatants were assayed by placing them in a dosimeter versus theirrespective pellets. The values are shown below in Table 4.

                  TABLE 4                                                         ______________________________________                                        Compound          Supernatant                                                                             Pellet                                            ______________________________________                                        orosomucoid       90%       10%                                                 biotin-oroso   7.7%  92.%                                                     asialoorosomucoid 92%   8.0%                                                  biotin-asialo 10% 90%                                                       ______________________________________                                    

C. Protein-Streptavidin Binding in vivo. Biotin-asialoorosomucoid wasevaluated for the ability to couple with circulating LU-10-StrAvconjugate in vivo and to remove it from the blood. Female BALB/c mice(20-25 g) were injected i.v. with 200 μg LU-10-StrAv conjugate. Clearingagent (200 μl PBS--group 1; 400 μg non-biotinylatedasialoorosomucoid--group 2; 400 μg biotinylated asialoorosomucoid--group3; and 200 μg biotinylated asialoorosomucoid--group 4) was administeredat 25 hours following conjugate administration. A fifth group receivedPIP-I-131-LU-10-StrAv conjugate which had been saturated prior toinjection with biotin--group 5. The 400 μg dose constituted a 10:1 molarexcess of clearing agent over the initial dose of LU-10-StrAv conjugate,while the 200 μg dose constituted a 5:1 molar excess. The saturatedPIP-I-131-LU-10-StrAv conjugate was produced by addition of a 10-foldmolar excess of D-biotin to 2 mg of LU-10-StrAv followed by sizeexclusion purification on a G-25 PD-10 column.

Three mice from each group were serially bled, as described above, at0.17, 1, 4 and 25 hours (pre-injection of clearing agent), as well as at27, 28, 47, 70 and 90 hours. Two additional animals from each group weresacrificed at 2 hours post-clearing agent administration and limitedbiodistributions were performed.

The blood clearance data are shown in FIG. 7. These data indicate thatcirculating LU-10-StrAv radioactivity in groups 3 and 4 was rapidly andsignificantly reduced, in comparison to those values obtained in thecontrol groups 1, 2 and 5. Absolute reduction in circulatingantibody-streptavidin conjugate was approximately 75% when compared tocontrols.

Biodistribution data are shown in tabular form in FIG. 8. Thebiodistribution data show reduced levels of conjugate for groups 3 and 4in all tissues except the liver, kidney and intestine, which isconsistent with the processing and excretion of radiolabel associatedwith the conjugate after complexation with biotinylatedasialoorosomucoid.

Furthermore, residual circulating conjugate was obtained from serumsamples by cardiac puncture (with the assays conducted in serum+PBS) andanalyzed for the ability to bind biotin (immobilized biotin on agarosebeads), an indicator of functional streptavidin remaining in the serum.Group 1 animal serum showed conjugate radiolabel bound about 80% toimmobilized biotin. Correcting the residual circulating radiolabelvalues by multiplying the remaining percent injected dose (at 2 hoursafter clearing agent administration) by the remaining percent able tobind immobilize biotin (the amount of remaining functional conjugate)leads to the graph shown in FIG. 9. Administration of 200 μgbiotinylated asialoorosomucoid resulted in a 50-fold reduction in serumbiotin-binding capacity and, in preliminary studies in tumored animals,has not exhibited cross-linking and removal of prelocalized LU-10-StrAvconjugate from the tumor. Removal of circulating targetingmoiety-anti-ligand without diminishing biotin-binding capacity at targetcell sites, coupled with an increased radiation dose to the tumorresulting from an increase in the amount of targeting moiety-anti-ligandadministered, results in both increased absolute rad dose to tumor anddiminished toxicity to non-tumor cells, compared to what is currentlyachievable using conventional radioimmunotherapy.

A subsequent experiment was executed to evaluate lower doses ofasialoorosomucoid-biotin. In the same animal model, doses of 50, 20 and10 μg asialoorosomucoid-biotin were injected at 24 hours followingadministration of the LU-10-StrAv conjugate. Data from animals seriallybled are shown in FIG. 10, and data from animals sacrificed two hoursafter clearing agent administration are shown in FIGS. 11A (bloodclearance) and 11B (serum biotin-binding), respectively. Doses of 50 and20 μg asialoorosomucoid-biotin effectively reduced circulatingLU-10-StrAv conjugate levels by about 65% (FIG. 11A) and, aftercorrection for binding to immobilized biotin, left only 3% of theinjected dose in circulation that possessed biotin-binding capacity,compared with about 25% of the injected dose in control animals (FIG.11B). Even at low doses (approaching 1:1 stoichiometry with circulatingLU-10-StrAv conjugate), asialoorosomucoid-biotin was highly effective atreducing blood levels of circulating streptavidin-containing conjugateby an in vivo complexation that was dependent upon biotin-avidininteraction.

EXAMPLE XIV Tumor Uptake of PIP-Biocytin

PIP-Biocytin, as prepared and described in Example VII above, was testedto determine the fate thereof in vivo. The following data are based onexperimentation with tumored nude mice (100 mg LS-180 tumor xenograftsimplanted subcutaneously 7 days prior to study) that received, at time0, 200 μg of I-125 labeled NR-LU-10-Streptavidin conjugate (950 pmol),as discussed in Example XI above. At 24 hours, the mice received an i.v.injection of PIP-I-131-biocytin (40 μCi) and an amount of cold carrierPIP-I-127 biocytin corresponding to doses of 42 μg (69,767 pmol), 21 μg(34,884 pmol), 5.7 μg (9468 pmol), 2.85 μg (4734 pmol) or 0.5 μg (830pmol). Tumors were excised and counted for radioactivity 4 hours afterPIP-biocytin injection.

The three highest doses produced PIP-biocytin tumor localizations ofabout 600 pmol/g. Histology conducted on tissues receiving the twohighest doses indicated that saturation of tumor-bound streptavidin wasachieved. Equivalent tumor localization observed at the 5.7 μg dose isindicative of streptavidin saturation as well. In contrast, the twolowest doses produced lower absolute tumor localization of PIP-biocytin,despite equivalent localization of NR-LU-10-Streptavidin conjugate(tumors in all groups averaged about 40% ID/G for the conjugate).

The lowest dose group (0.5 μg) exhibited high efficiency tumor deliveryof PIP-I-131-biocytin, which efficiency increased over time. A peakuptake of 85.0% ID/G was observed at the 120 hour time point (96 hoursafter administration of PIP-biocytin). Also, the absolute amount ofPIP-biocytin, in terms of % ID, showed a continual increase in the tumorover all of the sampled time points. The decrease in uptake on a % ID/Gbasis at the 168 hour time point resulted from significant growth of thetumors between the 120 and 168 hour time points.

In addition, the co-localization of NR-LU-10-Streptavidin conjugate(LU-10-StrAv) and the subsequently administered PIP-Biocytin at the sametumors over time was examined. The localization of radioactivity attumors by PIP-biocytin exhibited a pattern of uptake and retention thatdiffered from that of the antibody-streptavidin conjugate (LU-10-StrAv).LU-10-StrAv exhibited a characteristic tumor uptake pattern that isequivalent to historical studies of native NR-LU-10 antibody, reaching apeak value of 40% ID/G between 24 and 48 hours after administration. Incontrast, the PIP-Biocytin exhibited an initial rapid accretion in thetumor, reaching levels greater than those of LU-10-StrAv by 24 hoursafter PIP-Biocytin administration. Moreover, the localization ofPIP-Biocytin continued to increase out to 96 hours, when theconcentration of radioactivity associated with the conjugate has begunto decrease. The slightly greater amounts of circulating PIP-Biocytincompared to LU-10-StrAv at these time points appeared insufficient toaccount for this phenomenon.

The ratio of PIP-Biocytin to LU-10-StrAv in the tumor increasedcontinually during the experiment, while the ratio in the blooddecreased continually. This observation is consistent with a processinvolving continual binding of targeting moiety-containing conjugate(with PIP-Biocytin bound to it) from the blood to the tumor, withsubsequent differential processing of the PIP-Biocytin and theconjugate. Since radiolabel associated with the streptavidin conjugatecomponent (compared to radiolabel associated with the targeting moiety)has shown increased retention in organs of metabolic processing,PIP-Biocytin associated with the streptavidin appears to be selectivelyretained by the tumor cells. Because radiolabel is retained at targetcell sites, a greater accumulation of radioactivity at those sitesresults.

The AUC_(tumor) /AUC_(blood) for PIP-Biocytin is over twice that of theconjugate (4.27 compared to 1.95, where AUC means "area under thecurve"). Further, the absolute AUC_(tumor) for PIP-Biocytin is nearlytwice that of the conjugate (9220 compared to 4629). Consequently, anincrease in radiation dose to tumor was achieved.

EXAMPLE XV Clearing Agent Evaluation Experimentation

The following experiments conducted on non-tumor-bearing mice wereconducted using female BALB/c mice (20-25 g). For tumor-bearing miceexperimentation, female nude mice were injected subcutaneously withLS-180 tumor cells, and, after 7 d, the mice displayed 50-100 mg tumorxenografts. The monoclonal antibody used in these experiments wasNR-LU-10. When radiolabeled, the NR-LU-10-streptavidin conjugate wasradiolabeled with I-125 using procedures described herein. Whenradiolabeled, PIP-biocytin was labeled with I-131 or I-125 usingprocedures described herein.

A. Utility of Asialoorosomucoid-Biotin (AO-Bt) in Reducing CirculatingRadioactivity from a Subseguently Administered Radiolabeled BiotinLigand. Mice bearing LS-180 colon tumor xenografts were injected with200 micrograms NR-LU-10 antibody-streptavidin (MAb-StrAv) conjugate attime 0, which was allowed to prelocalize to tumor for 22 hours. At thattime, 20 micrograms of AO-Bt was administered to one group of animals.Two hours later, 90 micrograms of a radioisotope-bearing,ligand-containing small molecule (PIP-biotin-dextran prepared asdiscussed in part B hereof) was administered to this group of mice andalso to a group which had not received AO-Bt. The results of thisexperiment with respect to radiolabel uptake in tumor and clearance fromthe blood indicated that tumor-targeting of the radiolabeledbiotin-containing conjugate was retained while blood clearance wasenhanced, leading to an overall improvement in amount delivered totarget/amount located in serum. The AUC tumor/AUC blood with clearingagent was 6.87, while AUC tumor/AUC blood without clearing agent was4.45. Blood clearance of the circulating MAb-StrAv conjugate wasenhanced with the use of clearing agent. The clearing agent wasradiolabeled in a separate group of animals and found to bind directlyto tumor at very low levels (1.7 pmol/g at a dose of 488 total pmoles(0.35% ID/g), indicating that it does not significantly compromise theability of tumor-bound MAb-StrAv to bind subsequently administeredradiolabeled ligand.

B. Preparation Protocol for PIP-Biotin-Dextran. A solution of 3.0 mgbiotin-dextran, lysine fixable (BDLF, available from Sigma Chemical Co.,St. Louis, Mo., 70,000 dalton molecular weight with approximately 18biotins/molecule) in 0.3 ml PBS and 0.15 ml 1 M sodium carbonate, pH9.25, was added to a dried residue (1.87 mCi) of N-succinimidylp-I-125-iodobenzoate prepared in accordance with Wilbur, et al., J.Nucl. Med., 30: 216-226, 1989.

C. Dosing Optimization of AO-Bt. Tumored mice receiving StrAv-MAb asabove, were injected with increasing doses of AO-Bt (0 micrograms, 20micrograms, 50 micrograms, 100 micrograms and 200 micrograms). Tumoruptake of I-131-PIP-biocytin (5.7 micrograms, administered 2 hours afterAO-Bt administration) was examined. Increasing doses of AO-Bt had noeffect on tumor localization of MAb-StrAv. Data obtained 44 hours afterAO-Bt administration showed the same lack of effect. This data indicatesthat AO-Bt dose not cross-link and internalize MAb-StrAv on the tumorsurface, as had been noted for avidin administered followingbiotinylated antibody.

PIP-biocytin tumor localization was inhibited at higher doses of AO-Bt.This effect is most likely due to reprocessing and distribution to tumorof biotin used to derivatize AO-Bt. Optimal tumor to blood ratios (%injected dose of radiolabeled ligand/gram weight of tumor divided by %injected dose of radioligand/gram weight of blood were achieved at the50 microgram dose of AO-Bt. Biodistributions conducted followingcompletion of the protocols employing a 50 microgram AO-Bt dose revealedlow retention of radiolabel in all non-target tissues (1.2 pmol/g inblood; 3.5 pmol/gram in tail; 1.0 pmol/g in lung; 2.2 pmol/g in liver;1.0 pmol/g is spleen; 7.0 pmol/g in stomach; 2.7 pmol/g in kidney; and7.7 pmol/g in intestine). With 99.3 pmol/g in tumor, these resultsindicate effective decoupling of the PIP-biocytin biodistribution fromthat of the MAb-StrAv at all sites except tumor. This decouplingoccurred at all clearing agent doses in excess of 50 micrograms as well.Decreases in tumor localization of PIP-biocytin was the significantresult of administering clearing agent doses in excess of 50 micrograms.In addition, the amount of PIP-biocytin in non-target tissues 44 hoursafter administration was identical to localization resulting fromadministration of PIP-biocytin alone (except for tumor, where negligibleaccretion was seen when PIP-biocytin was administered alone), indicatingeffective decoupling.

D. Further Investigation of Optimal Clearing Agent Dose. Tumored miceinjected with MAb-StrAv at time 0 as above; 50 micrograms of AO-Bt attime 22 hours; and 545 microcuries of I-131-PIP-biocytin at time 25hours. Whole body radiation was measured and compared to that of animalsthat had not received clearing agent. 50 micrograms of AO-Bt wasefficient in allowing the injected radioactivity to clear from theanimals unimpeded by binding to circulating MAb-StrAv conjugate. Tumoruptake of I-131-PIP-biocytin was preserved at the 50 microgram clearingagent dose, with AUC tumor/AUC blood of 30:1 which is approximately15-fold better than the AUC tumor/AUC blood achieved in conventionalantibody-radioisotope therapy using this model.

E. Galactose- and Biotin-Derivatization of Human Serum Albumin (HSA).HSA was evaluated because it exhibits the advantages of being bothinexpensive and non-immunogenic. HSA was derivatized with varying levelsof biotin (1-about 9 biotins/molecule) via analogous chemistry to thatpreviously described with respect to AO. More specifically, to asolution of HSA available from Sigma Chemical Co. (5-10 mg/ml in PBS)was added 10% v/v 0.5 M sodium borate buffer, pH 8.5, followed bydropwise addition of a DMSO solution of NHS-LC-biotin (Sigma ChemicalCo.) to the stirred solution at the desired molar offering (relativemolar equivalents of reactants). The final percent DMSO in the reactionmixture should not exceed 5%. After stirring for 1 hour at roomtemperature, the reaction was complete. A 90% incorporation efficiencyfor biotin on HSA was generally observed. As a result, if 3 molarequivalences of the NHS ester of LC-biotin was introduced, about 2.7biotins per HSA molecule were obtained. Unreacted biotin reagent wasremoved from the biotin-derivatized HSA using G-25 size exclusionchromatography. Alternatively, the crude material may be directlygalactosylated. The same chemistry is applicable for biotinylatingnon-previously biotinylated dextran.

HSA-biotin was then derivatized with from 12 to 15 galactoses/molecule.Galactose derivatization of the biotinylated HSA was performed accordingto the procedure of Lee, et al., Biochemistry, 15: 3956, 1976. Morespecifically, a 0.1 M methanolic solution ofcyanomethyl-2,3,4,6-tetra-O-acetyl-1-thio-D-galactopyranoside wasprepared and reacted with a 10% v/v 0.1 M NaOMe in methanol for 12 hoursto generate the reactive galactosyl thioimidate. The galactosylation ofbiotinylated HSA began by initial evaporation of the anhydrous methanolfrom a 300 fold molar excess of reactive thioimidate. Biotinylated HSAin PBS, buffered with 10% v/v 0.5 M sodium borate, was added to the oilyresidue. After stirring at room temperature for 2 hours, the mixture wasstored at 4° C. for 12 hours. The galactosylated HSA-biotin was thenpurified by G-25 size exclusion chromatography or by buffer exchange toyield the desired product. The same chemistry is exploitable togalactosylating dextran. The incorporation efficiency of galactose onHSA is approximately 10%.

70 micrograms of Galactose-HSA-Biotin (G-HSA-B), with 12-15 galactoseresidues and 9 biotins, was administered to mice which had beenadministered 200 micrograms of StrAv-MAb or 200 microliters of PBS 24hours earlier. Results indicated that G-HSA-B is effective in removingStrAv-MAb from circulation. Also, the pharmacokinetics of G-HSA-B isunperturbed and rapid in the presence or absence of circulatingMAb-StrAv.

F. Non-Protein Clearing Agent. A commercially available form of dextran,molecular weight of 70,000 daltons, pre-derivatized with approximately18 biotins/molecule and having an equivalent number of free primaryamines was studied. The primary amine moieties were derivatized with agalactosylating reagent, substantially in accordance with the proceduretherefor described above in the discussion of HSA-based clearing agents,at a level of about 9 galactoses/molecule. The molar equivalenceoffering ratio of galactose to HSA was about 300:1, with about one-thirdof the galactose being converted to active form. 40 Micrograms ofgalactose-dextran-biotin (GAL-DEX-BT) was then injected i.v. into onegroup of mice which had received 200 micrograms MAb-StrAv conjugateintravenously 24 hours earlier, while 80 micrograms of GAL-DEX-BT wasinjected into other such mice. GAL-DEX-BT was rapid and efficient atclearing StrAv-MAb conjugate, removing over 66% of circulating conjugatein less than 4 hours after clearing agent administration. An equivalenteffect was seen at both clearing agent doses, which correspond to 1.6(40 micrograms) and 3.2 (80 micrograms) times the stoichiometric amountof circulating StrAv conjugate present.

G. Dose Ranging for G-HSA-B Clearing Agent. Dose ranging studiesfollowed the following basic format:

200 micrograms MAb-StrAv conjugate administered;

24 hours later, clearing agent administered; and

2 hours later, 5.7 micrograms PIP-biocytin administered.

Dose ranging studies were performed with the G-HSA-B clearing agent,starting with a loading of 9 biotins per molecule and 12-15 galactoseresidues per molecule. Doses of 20, 40, 70 and 120 micrograms wereadministered 24 hours after a 200 microgram dose of MAb-StrAv conjugate.The clearing agent administrations were followed 2 hours later byadministration of 5.7 micrograms of I-131-PIP-biocytin. Tumor uptake andblood retention of PIP-biocytin was examined 44 hours afteradministration thereof (46 hours after clearing agent administration).The results showed that a nadir in blood retention of PIP-biocytin wasachieved by all doses greater than or equal to 40 micrograms of G-HSA-B.A clear, dose-dependent decrease in tumor binding of PIP-biocytin ateach increasing dose of G-HSA-B was present, however. Since nodose-dependent effect on the localization of MAb-StrAv conjugate at thetumor was observed, this data was interpreted as being indicative ofrelatively higher blocking of tumor-associated MAb-StrAv conjugate bythe release of biotin from catabolized clearing agent. Similar resultsto those described earlier for the asialoorosomucoid clearing agentregarding plots of tumor/blood ratio were found with respect to G-HSA-B,in that an optimal balance between blood clearance and tumor retentionoccurred around the 40 microgram dose.

Because of the relatively large molar amounts of biotin that could bereleased by this clearing agent at higher doses, studies were undertakento evaluate the effect of lower levels of biotinylation on theeffectiveness of the clearing agent. G-HSA-B, derivatized with either 9,5 or 2 biotins/molecule, was able to clear MAb-StrAv conjugate fromblood at equal protein doses of clearing agent. All levels ofbiotinylation yielded effective, rapid clearance of MAb-StrAv fromblood.

Comparison of these 9-, 5-, and 2-biotin-derivatized clearing agentswith a single biotin G-HSA-B clearing agent was carried out in tumoredmice, employing a 60 microgram dose of each clearing agent. Thisexperiment showed each clearing agent to be substantially equallyeffective in blood clearance and tumor retention of MAb-StrAv conjugate2 hours after clearing agent administration. The G-HSA-B with a singlebiotin was examined for the ability to reduce binding of a subsequentlyadministered biotinylated small molecule (PIP-biocytin) in blood, whilepreserving tumor binding of PIP-biocytin to prelocalized MAb-StrAvconjugate. Measured at 44 hours following PIP-biocytin administration,tumor localization of both the MAb-StrAv conjugate and PIP-biocytin waswell preserved over a broad dose range of G-HSA-B with onebiotin/molecule (90 to 180 micrograms). A progressive decrease in bloodretention of PIP-biocytin was achieved by increasing doses of the singlebiotin G-HSA-B clearing agent, while tumor localization remainedessentially constant, indicating that this clearing agent, with a lowerlevel of biotinylation, is preferred. This preference arises because thesingle biotin G-HSA-B clearing agent is both effective at clearingMAb-StrAv over a broader range of doses (potentially eliminating theneed for patient-to-patient titration of optimal dose) and appears torelease less competing biotin into the systemic circulation than thesame agent having a higher biotin loading level.

Another way in which to decrease the effect of clearing agent-releasedbiotin on active agent-biotin conjugate binding to prelocalizedtargeting moiety-streptavidin conjugate is to attach the protein orpolymer or other primary clearing agent component to biotin using aretention linker. A retention linker has a chemical structure that isresistant to agents that cleave peptide bonds and, optionally, becomesprotonated when localized to a catabolizing space, such as a lysosome.Preferred retention linkers of the present invention are short stringsof D-amino acids or small molecules having both of the characteristicsset forth above. An exemplary retention linker of the present inventionis cyanuric chloride, which may be interposed between an epsilon aminogroup of a lysine of a proteinaceous primary clearing agent componentand an amine moiety of a reduced and chemically altered biotin carboxymoiety (which has been discussed above) to form a compound of thestructure set forth below. ##STR11## When the compound shown above iscatabolized in a catabolizing space, the heterocyclic ring becomesprotonated. The ring protonation prevents the catabolite from exitingthe lysosome. In this manner, biotin catabolites containing theheterocyclic ring are restricted to the site(s) of catabolism and,therefore, do not compete with active-agent-biotin conjugate forprelocalized targeting moiety-streptavidin target sites.

Comparisons of tumor/blood localization of radiolabeled PIP-biocytinobserved in the G-HSA-B dose ranging studies showed that optimal tumorto background targeting was achieved over a broad dose range (90 to 180micrograms), with the results providing the expectation that even largerclearing agent doses would also be effective. Another key result of thedose ranging experimentation is that G-HSA-B with an average of only 1biotin per molecule is presumably only clearing the MAb-StrAv conjugatevia the Ashwell receptor mechanism only, because too few biotins arepresent to cause cross-linking and aggregation of MAb-StrAv conjugatesand clearing agents with such aggregates being cleared by thereticuloendothelial system.

H. Tumor Targeting Evaluation Using G-HSA-B. The protocol for thisexperiment was as follows:

Time 0: administer 400 micrograms MAb-StrAv conjugate;

Time 24 hours: administer 240 micrograms of G-HSA-B with one biotin and12-15 galactoses and Time 26 hours: administer 6 micrograms of ##STR12##Lu-177 is complexed with the DOTA chelate using known techniquestherefor, and the DOTA chelate is prepared in accordance with thefollowing procedure. N-methyl-glycine (trivial name sarcosine, availablefrom Sigma Chemical Co.) was condensed with biotin-NHS ester in DMF andtriethylamine to obtain N-methyl glycyl-biotin. N-methyl-glycyl biotinwas then activated with EDCI and NHS. The resultant NHS ester was notisolated and was condensed in situ with DOTA-aniline preparable usingknown techniques (e.g., McMurry et al., Bioconiugate Chem., 3: 108-117,1992) and excess pyridine. The reaction solution was heated at 60° C.for 10 minutes and then evaporated. The residue was purified bypreparative HPLC to give[(N-methyl-N-biotinyl)-N-glycyl]-aminobenzyl-DOTA.

1. Preparation of (N-methyl)glycyl biotin. DMF (8.0 ml) andtriethylamine (0.61 ml, 4.35 mmol) were added to solids N-methyl glycine(182 mg, 2.05 mmol) and N-hydroxy-succinimidyl biotin (500 mg, 1.46mmol). The mixture was heated for 1 hour in an oil bath at 85° C. duringwhich time the solids dissolved producing a clear and colorlesssolution. The solvents were then evaporated. The yellow oil residue wasacidified with glacial acetic acid, evaporated and chromatographed on a27 mm column packed with 50 g silica, eluting with 30% MeOH/EtOAc 1%HOAc to give the product as a white solid (383 mg) in 66% yield.

H-NMR (DMSO): 1.18-1.25 (m, 6H, (CH₂)₃), 2.15, 2.35 (2 t's, 2H, CH₂ CO),2.75 (m, 2H, SCH₂), 2.80, 3.00 (2 s's, 3H, NCH₃), 3.05-3.15 (m, 1H,SCH), 3.95, 4.05 (2 s's, 2H, CH₂ N), 4.15, 4.32 (2 m's, 2H, 2CHN's),6.35 (s, NH), 6.45 (s, NH).

2. Preparation of [(N-methyl-N-biotinyl)glycyl] aminobenzyl-DOTA.N-hydroxysuccinimide (10 mg, 0.08 mmol) and EDCI (15 mg, 6.08 mmol) wereadded to a solution of (N-methylglycyl biotin (24 mg, 0.08 mmol) in DMF(1.0 ml). The solution was stirred at 23° C. for 64 hours. Pyridine (0.8ml) and aminobenzyl-DOTA (20 mg, 0.04 mmol) were added. The mixture washeated in an oil bath at 63° C. for 10 minutes, then stirred at 23° C.for 4 hours. The solution was evaporated. The residue was purified bypreparative HPLC to give the product as an off white solid (8 mg, 0.01mmol) in 27% yield.

H-NMR (D₂ O): 1.30-1.80 (m, 6H), 2.40, 2.55 (2 t's, 2H, CH₂ CO),2.70-4.2 (complex multiplet), 4.35 (m, CHN), 4.55 (m, CHN), 7.30 (m, 2H,benzene hydrogens), 7.40 (m, 2H, benzene hydrogens).

Efficient delivery of the Lu-177-DOTA-biotin small molecule wasobserved, 20-25% injected dose/gram of tumor. These values areequivalent with the efficiency of the delivery of the MAb-StrAvconjugate. The AUC tumor/AUC blood obtained for this non-optimizedclearing agent dose was 300% greater than that achievable by comparabledirect MAb-radiolabel administration. Subsequent experimentation hasresulted in AUC tumor/AUC blood over 1000% greater than that achievableby comparable conventional MAb-radiolabel administration. In addition,the HSA-based clearing agent is expected to exhibit a low degree ofimmunogenicity in humans.

EXAMPLE XVI Palytoxin-Containing Conjugates

A. Palytoxin-mono-oxyacetyl-LC-biotin.

Trichloroethyl carbamate-NH-palytoxin (troc-NH-palytoxin).Trichloroethyl-chloroformate (available from Aldrich Chemical Co.,Milwaukee, Wis.) is added to a solution of palytoxin (available fromHawaii Biotechnology Group, Inc., Aiea, Hi.) in pyridine. The solutionis stirred at 23° C. for 6 hours, and the solvents are evaporated underreduced pressure. The residue is dissolved in water and washed with CH₂Cl₂. The aqueous fraction is lyophilized, and the product is purified byCM-Sephadex D-25 chromatography. (Trichloroethyl carbamate(Troc)-NH)-palytoxin-oxyacetyl-LC-biotin. 1.0 equivalent of sodiumhydride is added to a solution of troc-NH-palytoxin in DMF followed by1.0 equivalent of iodoacetyl-LC-biotin. The suspension is stirred at 23°C. for 24 hours, then quenched by the addition of water; acidified to pH7 by the addition of 0.1M HCl; and troc-deprotected (zinc in aqueous THFin a phosphate buffer, pH 5.5) to afford the final product.

B.Palytoxin-mono-oxo-N-acetamido-ethyldiamine-N'-(N-methyl)-glycyl-biotin.

N-methyl-glycyl-biotin. N-methyl-glycine (trivial name sarcosine,available from Sigma Chemical Co.) was condensed with biotin-NHS esterin DMF and triethylamine to obtain N-methyl glycyl-biotin.

More specifically, DMF (8.0 ml) and triethylamine (0.61 ml, 4.35 mmol)were added to solids N-methyl glycine (182 mg, 2.05 mmol) andN-hydroxy-succinimidyl biotin (500 mg, 1.46 mmol). The mixture washeated for 1 hour in an oil bath at 85° C. during which time the solidsdissolved producing a clear and colorless solution. The solvents werethen evaporated. The yellow oil residue was acidified with glacialacetic acid, evaporated and chromatographed on a 27 mm column packedwith 50 g silica, eluting with 30% MeOH/EtOAc 1% HOAc to give theproduct as a white solid (383 mg) in 66% yield.

H-NMR (DMSO): 1.18-1.25 (m, 6H, (CH₂)₃), 2.15, 2.35 (2 t's, 2H, CH₂ CO),2.75 (m, 2H, SCH₂), 2.80, 3.00 (2 s's, 3H, NCH₃), 3.05-3.15 (m, 1H,SCH), 3.95, 4.05 (2 s's, 2H, CH₂ N), 4.15, 4.32 (2 m's, 2H, 2CHN's),6.35 (s, NH), 6.45 (s, NH).

Palytoxin-mono-oxo-N-acetamido-ethyldiamine-N'-(N-methyl)-glycyl-biotin.N-methyl-glycyl-biotin is esterified in refluxing methanol containinggaseous HCl to give the methylester, methyl-(N-methyl)glycyl-biotin.This methyl ester is dissolved in ethylene diamine and stirred at 23° C.for 14 hours to afford biotinyl-N-methyl-glycyl-ethylene diaminemonoamide. The solvent (ethylene diamine) is evaporated under vacuum.The amino group of biotinyl-N-methyl-glycyl-ethylene diamine monoamideis acylated with N-hydroxysuccinimidyl iodoacetate (prepared fromiodoacetic acid available from Aldrich Chemical Co.,N-hydroxysuccinimide and dicyclohexylcarbodiimide (DCC)) in DMF withtriethylamine to form an acylated iodinated biotin derivative.Troc-NH-palytoxin prepared as set forth above is deprotonated with 1.0equivalents of sodium hydride and O-alkylated with the iodo-biotinderivative, N-(iodoacetyl)-ethylene-diamine-N'-(N-methyl)glycylbiotin,to afford the ether,N-(trichloroethoxycarbonyl)-palytoxin-mono-oxo-N-acetamido-ethylenediamine-N'-(N-methyl)glycylbiotin.The trichloro-carbamate group of the ether is cleaved with zinc inaqueous THF, pH 5.5-7.2, to afford the final product.

C. Carboxy-liberated palytoxin-N(Me)-glycyl-biotin.

The two amide bonds of Troc-amine protected palytoxin(troc-NH-palytoxin) are cleaved with peptidase (available from SigmaChemical Company), a compound that selectively cleaves amide bonds undermild conditions, at pH 7.1 and 37° C. to afford a palytoxin moleculewith a free carboxy group. This product is purified by preparativeHPLC--reverse phase C-18 chromatography. The free carboxy group isactivated with EDCI and coupled to biotinyl-N-methyl-glycyl-ethylenediamine monoamide to giveN-(trocNH)-palytoxinamido-ethylenediamine-N'-(N-methyl)-glycyl-biotin.The troc group is cleaved with zinc in aqueous THF to afford the finalproduct.

D. C-55 palytoxin-biotin conjugate.

Trichloroethyl carbamate-NH-palytoxin (troc-NH-palytoxin).Trichloroethyl-chloroformate (available from Aldrich Chemical Co.) isadded to a solution of palytoxin in pyridine. The solution is stirred at23° C. for 6 hours, and the solvents are evaporated under reducedpressure. The residue is dissolved in water and washed with CH₂ Cl₂. Theaqueous fraction is lyophilized, and the product is purified onCM-Sephadex D-25 chromatography.

Troc-NH-palytoxin-C₅₅ -NH-(CH₂)₅ -NH-biotin. To a solution containing1.0 equivalent of troc-NH-palytoxin and 1.0 equivalent ofbiotinylpentylamine (available from Pierce Chemical Co., Rockford, Ill.)in methanol (adjusted to pH 6 by addition of 5N HCl/methanol) is added1.0 equivalent of NaCNBH₃. This reaction mixture is stirred for 24 hoursat 23° C. to afford the biotinylated palytoxin product.

E. Palytoxin-streptavidin conjugation.

The amide bond at carbon 17 of Troc-amine protected palytoxin is cleavedwith peptidase (available from Sigma Chemical Company), a compound thatselectively cleaves amide bonds under mild conditions, at pH 7.1 and 37°C. to afford a palytoxin molecule with a free carboxy group. Thisproduct is purified by preparative HPLC--reverse phase C-18chromatography. The free carboxy group is activated with EDCI andcoupled to streptavidin (available from Sigma Chemical Company) to giveN-troc-palytoxinamido-streptavidin. The troc group is cleaved with zincin aqueous THF (>5:1 H₂ O:THF) to afford the final product.

EXAMPLE XVII Polymer-Ligand Conjugation

Polylysine (approximately 10,000 Dal. molecular weight, available fromSigma Chemical Co., St. Louis, Mo.) and dextran (lysine fixable,available from Sigma Chemical Co.) were derivitized with SPDP andpurified from unreacted SPDP using size exclusion chromatography (usinga PD-10 column available from Pharmacia, Piscataway, N.J.). Theresultant SPDP-derivitized adducts were reduced with DTT in pH 4.7 0.2 MNaOAc buffer to generate free reactive thiols. Reduced Tc-99m, generatedfrom stannous gluconate as described, for example, by Carlsson et al.,Biochem. J., 173: 723-737, 1978, was added. A 90% incorporation ofTc-99m was obtained for the polylysine adduct within 15 min, as measuredby ITLC. 96% of the radioactivity coeluted with the dextran using sizeexclusion (PD-10) chromatography. These results are indicative ofchelation.

EXAMPLE XVIII Synthesis of Toxin-Polymer-Ligand Conjugates

Trichothecene-Polymer-Ligand Conjugation. Experimentation involving theuse of a biotin-dextran-trichothecene conjugate in a pretargetingapproach included:

trace labeling using an I-125 PIP NHS ester of an available lysine of a70,000 dalton dextran molecule that had been biotinylated in accordancewith techniques discussed herein in Example XVII, yielding radiotaggeddextran biotin (represented as dextran*-biotin);

trichothecene drug conjugation to the remaining lysines using an NHSactivated trichothecene;

demonstration of binding of the drug-dextran*-biotin molecule toimmobilized avidin; and

assessment of serum clearance in mice.

Biotinylated dextran having a molecular weight of 70,000 daltons, with18 moles of biotin covalently bound thereto and 18 additional lysineepsilon amino groups, was purchased from Sigma Chemical Co. (St. Louis,Mo.). To radiolabel the material, 4 mC of I-125 PIP NHS ester ofspecific activity of 2200 mCi/mmole (New England Nuclear, Boston, Mass.)in acetonitrile in a 2 ml glass vial was blown down to dryness in anitrogen stream. 10 mg of biotinylated, lysine-derivatized dextranlyophilizate, reconstituted with 0.650 ml of 1.0M sodium borate, pH 9.0,was added to the iodinating compound and incubated at room temperaturefor 10 minutes. Following reaction, the radioiodinated dextran moietywas purified by size exclusion chromatography using a PD-10 column(Pharmacia, Uppsala, Sweden) equilibrated in phosphate buffered saline(i.e., 6.2M sodium phosphate, 150 mM NaCl, pH 7.2) containing 1%molecusol (Pharmatec, Alachua, Florida). The biotin-dextran* eluted fromthe column in the 2.4-4.8 ml fractions at a specific activity of 0.2mCi/mg and a concentration of 3.5 mg/ml.

To derivatize the biotin-dextran* with trichothecene, 0.9 ml ofbiotin-dextran* was diluted with 1.3 ml of sodium borate buffer, 0.3M,Ph 8.5, containing 1% molecusol followed by addition, with stirring of1.2 ml of DMSO containing 1.2 mg of2'-Desoxy-2'-alpha-(N-hydroxysuccinimidyl-3-dithiopropanoicacid)-Roridin A (i.e., 2:1 drug to available lysine molar ratio). Afterincubation for 1.5 hours at room temperature, each 1 ml aliquot ofreaction mixture was purified as noted above with a PD-10 columnequilibrated in PBS. Yields exceeded 85%.

To establish that the biotin-dextran*-trichothecene molecule was able tobind to avidin or streptavidin, 1 microgram of biotin-dextran* and 1microgram of biotin-dextran*-trichothecene were incubated for 15 minutesat room temperature with 1 unit of avidin insolubilized on agarose beads(Sigma Chemical Co., St. Louis, Mo.) in 0.2 ml of 0.2 M Pi buffer, pH6.3 containing 150 mM NaCl. Following this incubation, the percentradioactivity bound to the agarose beads was assessed after dilutionwith 1.4 ml buffer, centrifugation of the agarose suspension and threewashings of the pellets with 1.4 ml buffer. 100% binding was observedfor both biotin-dextran* and biotin-dextran*-trichothecene.

Serum clearance studies of biotin-dextran* andbiotin-dextran*-trichothecene were also performed in Balb C mice. Serialblood samplings revealed that he two molecules exhibited substantiallysimilar serum clearance upon injection of 2 μCi thereof.

Kits containing one or more of the components described above are alsocontemplated. For instance, radiohalogenated biotin may be provided in asterile container for use in pretargeting procedures. A chelate-biotinconjugate provided in a sterile container is suitable forradiometallation by the consumer; such kits would be particularlyamenable for use in pretargeting protocols. Alternatively,radiohalogenated biotin and a chelate-biotin conjugate may be vialed ina non-sterile condition for use as a research reagent.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

What is claimed is:
 1. A method of increasing localization of a cytokineat a target cell site within a mammalian recipient, which methodcomprises:administering to the recipient a first conjugate comprising atargeting moiety and streptavidin; and subsequently administering to therecipient a second conjugate comprising a cytokine and biotin, whereinthe method further comprises reducing the recipient's endogenous biotinlevel prior to, concurrently with or following administration of thefirst conjugate.
 2. A method of increasing localization of a cytokine ata target cell site within a mammalian recipient, which methodcomprises:administering to the recipient a first conjugate comprising atargeting moiety and streptavidin; and subsequently administering to therecipient a second conjugate comprising dextran-TNF-biotin, and furthermay comprise a cleavable linker which provides for the release of tumornecrosis factor at the target cell site.
 3. The method of claim 1,wherein endogenous biotin is reduced by administering high dosages ofthe targeting moiety-avidin or streptavidin conjugate, by pretreatmentwith an amount of avidin or streptavidin sufficient to bindsubstantially all endogenous biotin, by placing the recipient on abiotin-free diet prior to treatment, or by the administration of oral,non-absorbable antibiotics which suppress biotin.